Prognostic and predictive gene signature for non-small cell lung cancer and adjuvant chemotherapy

ABSTRACT

The application provides methods of prognosing and classifying lung cancer patients into poor survival groups or good survival groups and for determining the benefit of adjuvant chemotherapy by way of a multigene signature. The application also includes kits and computer products for use in the methods of the application.

PRIORITY

This application claims the benefit of U.S. Application No. 61/356,516, filed Jun. 18, 2010, which is hereby incorporated by reference in its entirety.

FIELD

The application relates to compositions and methods for prognosing and classifying non-small cell lung cancer and for determining the benefit of adjuvant chemotherapy.

BACKGROUND OF THE INVENTION

In North America, lung cancer is the leading cancer in males and the leading cause of cancer deaths in both males and females¹. Non-small cell lung cancer (NSCLC) represents 80% of all lung cancers and has an overall 5-year survival rate of only 16% ¹. Tumor stage is the primary determinant for treatment selection for NSCLC patients. Recent clinical trials have led to the adoption of adjuvant cisplatin-based chemotherapy in early stage NSCLC patients (Stages B-MA). The 5-year survival advantage conferred by adjuvant chemotherapy in recent trials are 4% in the International Adjuvant Lung Trial (IALT) involving 1,867 stage I-III patients², 15% in the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) BR.10 Trial involving 483 stage IB-II patients³, and 9% in the Adjuvant Navelbine International Trialist Association (ANITA) trial involving 840 stage IB-IIIA patients⁴. Pre-planned stratification analysis in the later two trials showed no significant survival benefit for stage IB patients^(3, 4). This was also demonstrated in the Cancer and Leukemia Group (CALGB) Trial 9633 that tested the benefit of chemotherapy on 344 stage IB patients receiving carboplatin and paclitaxel or observation⁵. Although initially presented in 2004 as a positive trial, recent survival analyses show no significant survival advantage with chemotherapy for either disease-free survival (HR=0.80, p=0.065) or overall survival (HR=0.83, p=0.12)⁵. In an attempt to draw an overall conclusion regarding the effectiveness of adjuvant cisplatin-based chemotherapy, the Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis⁶ was conducted which synthesized information from the 5 largest published, cisplatin-based trials that did not administer concurrent thoracic radiation [Adjuvant Lung Project Italy (ALPI)⁷, Big Lung Trial (BLT)⁸, IALT², BR.10³, and ANITA⁹]. The study found a 5.3% absolute survival advantage at 5-year (HR=0.89, 95% Cl 0.82-0.96, p=0.004). However, stratified analysis by stage showed that the stage iB patients did not benefit significantly from cisplatin treatment (HR=0.92, 95% Cl 0.78-1.10). Moreover, a detriment for chemotherapy was suggested in stage iA patients (HR=1.41, 95% Cl 0.96-2.09)⁶. Therefore, the current standard of treatment for patients with stage I NSCLC remains surgical resection alone. However, 30 to 40 percent of these stage I patients are expected to relapse after the initial surgery^(10, 11), indicating that a subgroup of these patients might benefit from adjuvant chemotherapy.

The lack of consistent prognostic molecular markers for early stage NSCLC patients led to attempts to identify novel gene expression signatures using genome wide microarray platforms. Such multi-gene signatures might be stronger than individual genes to predict poor prognosis and poor prognostic patients could potentially benefit from adjuvant therapies. Previous microarray studies have identified prognostic signatures that demonstrated minimal overlaps in the gene sets.¹²⁻²⁰ While only one of the early studies involved secondary signature validation in independent datasets¹², all recently reported signatures were tested for validation^(13-16, 20). Nevertheless, lack of direct overlaps between signatures remains. One of the potential confounding factors is that signatures were derived from patients operated at single institutions, which may introduce biases.

SUMMARY OF THE INVENTION

As discussed in the Background section, certain patients suffering from NSCLC benefit from adjuvant chemotherapy. Attempts to identify systematically patient subpopulations in which adjuvant therapy would lead to increased survival or improve patient prognosis have generally failed. Efforts to assemble prognostic molecular markers have yielded various non-overlapping gene sets but have fallen short of establishing a gene signature with a minimal set of genes that is predictive regardless of the form of NSCLC (eg. adenocarcinoma or squamous cell carcinoma) or stage, and serves as a reliable classifier for adjuvant therapy benefit.

As will be discussed in more detail below, a set of fifteen genes were previously identified by microarray analysis whose expression level is useful in the prognosis of survival outcome and diagnosis of adjuvant therapy benefit. The prognostic and diagnostic value of the 15-gene set was verified by validation against independent data sets. In migrating this signature to a qPCR-based platform, it was discovered that a fewer number of genes can provide essentially the same predictive value, including a 13-gene signature. In various embodiments of the invention, the present disclosure provides methods and kits useful for obtaining and utilizing expression information for the fourteen or fewer genes to obtain prognostic and diagnostic information for a patient with NSCLC. In some embodiments, the invention provides methods and kits useful for obtaining and utilizing expression information of at least 5 of the 14 genes.

In another aspect of the disclosure, it is shown that the genes maintain their predictive value when moved from microarray detection platform to quantitative PCR, and are applicable to both fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) tissue samples.

The methods of the present disclosure generally involve obtaining from a patient relative expression data, at the DNA, messenger RNA (mRNA), or protein level, for each of the genes and micro RNAs (miRNAs) regulating those genes, included in the set. In some embodiments, the present disclosure involves processing the data and comparing the resulting information to one or more reference values. Relative expression levels are expression data normalized according to techniques known to those skilled in the art. Expression data may be normalized with respect to one or more genes with invariant expression, such as “housekeeping” genes. In some embodiments, expression data may be processed using standard techniques, such as transformation to a z-score, and/or software tools, such as RMAexpress v0.3.

In one aspect, the invention provides a method for preparing a gene expression profile indicative of response to adjuvant chemotherapy for NSCLC. The method comprises determining the level of expression of at least five genes from Table 4A. Table 4A discloses 13 genes shown herein to maintain the predictive capacity of a 15 gene signature identified by a microarray detection format. The gene expression profile may be prepared from a fresh frozen tumor specimen or a FFPE specimen, and may be determined by quantitative PCR, or other amplification detection platform, which as shown herein is sufficient for maintaining the predictive capacity. In various embodiments, the gene expression profile does not include expression levels for MLANA and/or MYT1L, and may be normalized based on the expression level of one or more additional genes, such as one or more of BAT1, TBP, PP1A, and GUSB. Exemplary target sequences, primer sequences, and probe sequences for preparing expression profiles are further described herein.

In another aspect, the invention provides a method for predicting the benefit of adjuvant chemotherapy for a patient having non-small cell lung cancer. The method comprises determining a gene expression profile that includes the level of expression of from 5 to 14 genes each indicative of survival in a NSCLC population, with the genes being listed in Table 3. Preferably, at least 5 genes (e.g., 5, 6, 7, 8, 9, 10, 11, 12, or 13 genes) are listed in Table 4A. The gene expression profile is prepared from expression data obtained from fresh frozen or FFPE tumor tissue samples using a quantitative PCR detection platform. The profile is then classified to predict whether the patient will benefit from adjuvant chemotherapy, as described herein. For example, the profile may be classified by analyzing the gene expression levels of the 5 to 14 genes in connection with a classifier algorithm. The classifier algorithm may classify samples into a high risk group that is likely to benefit from adjuvant chemotherapy, or a low risk group where adjuvant chemotherapy is less likely to benefit the patient.

In a third aspect, a multi-gene signature is provided for prognosing or classifying patients with lung cancer (e.g., NSCLC). In some embodiments, a five to fourteen-gene signature is provided, comprising reference values for each of the five to fourteen different genes based on relative expression data for each gene from a historical data set with a known outcome, such as good or poor survival, and/or known treatment, such as adjuvant chemotherapy. In one embodiment, four reference values are provided for each of the five to thirteen genes listed in Table 4A. In one embodiment, the reference values for each of the five to thirteen genes are principal component values, such as those set forth in Table 10 for example.

In some embodiments, a fourteen-gene signature comprises reference values for each of fourteen different genes based on relative expression data for each gene from a historical data set with a known outcome and/or known treatment. In some embodiments, reference values are provided for one gene in addition to those listed in Table 4A, and the gene is selected from those listed in Table 3. In some embodiments, a single reference value for each gene is provided.

In one aspect, relative expression data from a patient are combined with the gene-specific reference values on a gene-by-gene basis for each of the five to fourteen genes to generate a test value which allows prognosis or therapy recommendation. In some embodiments, relative expression data are subjected to an algorithm that yields a single test value, or combined score, which is then compared to a control value obtained from the historical expression data for a patient or pool of patients. In some embodiments, the control value is a numerical threshold for predicting outcomes, for example good and poor outcome, or making therapy recommendations, for example adjuvant therapy in addition to surgical resection or surgical resection alone. In some embodiments, a test value or combined score greater than the control value is predictive, for example, of high risk (poor outcome) or benefit from adjuvant therapy, whereas a combined score falling below the control value is predictive, for example, of low risk (good outcome) or lack of benefit from adjuvant therapy.

In one embodiment, the combined score is calculated from relative expression data multiplied by reference values, determined from historical data, for each gene. Accordingly, the combined score may be calculated using the algorithm of Formula I below:

Combined score=0.557×PC1+0.328×PC2+0.43×PC3+0.335×PC4

Where PC1 is the sum of the relative expression level for each gene in a multi-gene signature multiplied by a first principal component for each gene in the multi-gene signature, PC2 is the sum of the relative expression level for each gene multiplied by a second principal component for each gene, PC3 is the sum of the relative expression level for each gene multiplied by a third principal component for each gene, and PC4 is the sum of the relative expression level for each gene multiplied by a fourth principal component for each gene. In some embodiments, the combined score is referred to as a risk score. A risk score for a subject can be calculated by applying Formula I to relative expression data from a test sample obtained from the subject.

In some embodiments, PC1 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a first principal component for each gene, respectively, as set forth in Table 10; PC2 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a second principal component for each gene, respectively, as set forth in Table 10; PC3 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a third principal component for each gene, respectively, as set forth in Table 10; and PC4 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a fourth principal component for each gene, respectively, as set forth in Table 10.

The present disclosure provides a gene signature that is prognostic for survival as well as predictive for benefit from adjuvant chemotherapy.

Accordingly in one embodiment, the application provides a method of prognosing or classifying a subject with non-small cell lung cancer comprising the steps:

-   -   a. determining the expression of thirteen biomarkers in a test         sample from the subject, wherein the biomarkers correspond to         genes in Table 4A, and     -   b. comparing the expression of the thirteen biomarkers in the         test sample with expression of the thirteen biomarkers in a         control sample,         wherein a difference or a similarity in the expression of the         thirteen biomarkers between the control and the test sample is         used to prognose or classify the subject with NSCLC into a poor         survival group or a good survival group.

In an aspect, the application provides a method of predicting prognosis in a subject with non-small cell lung cancer comprising the steps:

-   -   a. obtaining a subject biomarker expression profile in a sample         of the subject;     -   b. obtaining a biomarker reference expression profile associated         with a prognosis, wherein the subject biomarker expression         profile and the biomarker reference expression profile each have         thirteen values, each value representing the expression level of         a biomarker, wherein each biomarker corresponds to one gene in         Table 4A; and     -   c. selecting the biomarker reference expression profile most         similar to the subject biomarker expression profile, to thereby         predict a prognosis for the subject.

In another aspect, the prognoses and classifying methods of the application can be used to select treatment. For example, the methods can be used to select or identify subjects who might benefit from adjuvant chemotherapy. Accordingly, in one embodiment, the application provides a method of selecting a therapy for a subject with NSCLC, comprising the steps:

-   -   a. classifying the subject with NSCLC into a poor survival group         or a good survival group according to the method of the         application; and     -   b. selecting adjuvant chemotherapy for the poor survival group         or no adjuvant chemotherapy for the good survival group.

In another embodiment, the application provides a method of selecting a therapy for a subject with NSCLC, comprising the steps:

-   -   a. determining the expression of thirteen biomarkers in a test         sample from the subject, wherein the thirteen biomarkers         correspond to the thirteen genes in Table 4A;     -   b. comparing the expression of the thirteen biomarkers in the         test sample with the thirteen biomarkers in a control sample;     -   c. classifying the subject in a poor survival group or a good         survival group, wherein a difference or a similarity in the         expression of the thirteen biomarkers between the control sample         and the test sample is used to classify the subject into a poor         survival group or a good survival group;     -   d. selecting adjuvant chemotherapy if the subject is classified         in the poor survival group and selecting no adjuvant         chemotherapy if the subject is classified in the good survival         group.

Another aspect of the application provides compositions for use with the methods described herein.

The application also provides for kits used to prognose or classify a subject with NSCLC into a good survival group or a poor survival group or for selecting therapy for a subject with NSCLC that includes detection agents that can detect the expression products of the biomarkers.

In one aspect, the present disclosure provides kits useful for carrying out the diagnostic and prognostic tests described herein. The kits generally comprise reagents and compositions for obtaining relative expression data for from 5 to 14 genes from Table 3, and including at least 5 genes from Table 4 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, or 13 genes from Table 4A). In certain embodiments, the kits comprise reagents and compositions for obtaining relative expression levels of the genes described in Table 4A, and optionally, an additional gene selected from among those listed in Table 3. As will be recognized by the skilled artisans, the contents of the kits will depend upon the means used to obtain the relative expression information.

Kits may comprise a labeled compound or agent capable of detecting protein product(s) or nucleic acid sequence(s) in a sample and means for determining the amount of the protein or mRNA in the sample (e.g., an antibody which binds the protein or a fragment thereof, or an oligonucleotide probe which binds to DNA or mRNA encoding the protein). Kits can also include instructions for interpreting the results obtained using the kit.

In some embodiments, the kits are oligonucleotide-based kits, which may comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a marker protein or (2) a pair of primers useful for amplifying a marker nucleic acid molecule. Kits may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kits can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kits can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of a kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

In some embodiments, the kits are antibody-based kits, which may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a marker protein; and, optionally, (2) a second, different antibody which binds to either the protein or the first antibody and is conjugated to a detectable label.

A further aspect provides computer implemented products, computer readable mediums and computer systems that are useful for the methods described herein.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

FIG. 1(A,B) shows the derivation and testing of a prognostic signature;

FIG. 2(A-F) shows the survival outcome based on the 15-gene signature in training and test sets;

FIG. 3(A-H) shows a comparison of chemotherapy vs. observation in low and high risk patients with microarray data;

FIG. 4 shows a consort diagram for microarray study of BR. 10 patients;

FIG. 5 shows the effect of adjuvant chemotherapy in microarray profiled patients;

FIG. 6 shows the effect of microarray batch processing at 2 different times. The samples were profiled in 2 batches at 2 times (January 2004 and June 2005). Unsupervised clustering shows that the expression patterns of these two batches differed significantly with samples arrayed on January 2004 aggregated in cluster 1 (93%) and samples arrayed on June 2005 in cluster 2 (73%).

FIG. 7 shows the risk score for the 15-gene signature (“Risk Score”), a 14-gene signature that omits MYT1L (“No MYTL1”), a 14-gene signature that omits MLANA (“No MLANA”), and a 13-gene signature that omits both MYTL1 and MLANA (“Both Removed”). There was no significant difference between the 15 gene signature risk score and either 14-gene signature, from which either MYT1L or MLANA was removed (p=0.25 and p=0.28, respectively). There was also no significant difference between risk scores generated by the 15-gene and the 13-gene signatures—when both MYT1L and MLANA were removed (p=0.098).

FIG. 8 shows the process for evaluating the gene signature for RT-qPCR and with different probe chemistries.

FIG. 9 shows exemplary probe selection for ATP1B1.

FIG. 10(A-E) shows RT-qPCR on FFPE specimens, and assay efficiency.

FIG. 11(A-E) shows assay and selection of best performing reference gene.

FIG. 12(A,B) shows the correlation between frozen and FFPE samples.

FIG. 13 shows the correlation between qPCR and microarray-based risk scores.

DETAILED DESCRIPTION OF THE INVENTION

The application in various embodiments relates to a set of 13 or 14 biomarkers, and/or subsets thereof, that form a gene signature, and provides methods, compositions, computer implemented products, detection agents and kits for preparing gene expression profiles, and for prognosing or classifying a subject with non-small cell lung cancer (NSCLC) and for determining the benefit of adjuvant chemotherapy.

In these and other embodiments, the application relates to preparing gene expression profiles from fresh frozen or FFPE NSCLC tumor tissue samples by quantitative RT-PCR (RT-qPCR). Such gene expression profiles comprise the level of expression of from 5 to 14 (e.g., 13) genes that are correlate with survival in NSCLC, and such genes are listed in Tables 3 and 4A. The gene expression profiles may further be normalized using expression data from one or more normalization genes. The gene expression profiles may be used for classifying samples to predict the benefit of adjuvant chemotherapy.

The term “biomarker” as used herein refers to a gene that is differentially expressed in tumor tissue excised from individuals with non-small cell lung cancer (NSCLC) according to prognosis and is predictive of different survival outcomes and of the benefit of adjuvant chemotherapy. In some embodiments, a 13-gene signature comprises 13 biomarker genes listed in Table 4A, or a subset thereof. One optional additional biomarker for a 14-gene signature may be selected from the genes listed in Table 3.

Accordingly, one aspect of the invention is a method of prognosing or classifying a subject with non-small cell lung cancer, comprising the steps:

a. determining the expression of thirteen biomarkers in a test sample from the subject, wherein the biomarkers correspond to genes in Table 4A, and

b. comparing the expression of the thirteen biomarkers in the test sample with expression of the thirteen biomarkers in a control sample,

wherein a difference or a similarity in the expression of the thirteen biomarkers between the control and the test sample is used to prognose or classify the subject with NSCLC into a poor survival group or a good survival group.

In another aspect, the application provides a method of predicting prognosis in a subject with non-small cell lung cancer (NSCLC) comprising the steps:

a. obtaining a subject biomarker expression profile in a sample of the subject;

b. obtaining a biomarker reference expression profile associated with a prognosis, wherein the subject biomarker expression profile and the biomarker reference expression profile each have thirteen values, each value representing the expression level of a biomarker, wherein each biomarker corresponds to a gene in Table 4; and

c. selecting the biomarker reference expression profile most similar to the subject biomarker expression profile, to thereby predict a prognosis for the subject.

The term “reference expression profile” as used herein refers to the expression of the biomarkers or genes, such as those listed in Table 3 and Table 4A associated with a clinical outcome in a NSCLC patient. The reference expression profile comprises values (e.g., 5 to 14 values), each value representing the expression level of a biomarker, wherein each biomarker corresponds to one gene in Table 3 or Table 4A. The reference expression profile is identified using one or more samples comprising tumor tissue (NSCLC) wherein the expression is similar between related samples defining an outcome class or group such as poor survival or good survival and is different to unrelated samples defining a different outcome class such that the reference expression profile is associated with a particular clinical outcome. The reference expression profile is accordingly a reference profile of the expression of the genes in Table 3 or Table 4A (including the gene sets and subsets described herein), to which the subject expression levels of the corresponding genes in a patient sample (e.g., the gene expression profile) are compared in methods for determining or predicting clinical outcome.

As used herein, the term “control” refers to a specific value or dataset that can be used to prognose or classify the value e.g expression level or reference expression profile obtained from the test sample associated with an outcome class. In one embodiment, a dataset may be obtained from samples from a group of subjects known to have NSCLC and good survival outcome or known to have NSCLC and have poor survival outcome or known to have NSCLC and have benefited from adjuvant chemotherapy or known to have NSCLC and not have benefited from adjuvant chemotherapy. The expression data of the biomarkers in the dataset can be used to create a “control value” that is used in testing samples from new patients. A control value is obtained from the historical expression data for a patient or pool of patients with a known outcome. In some embodiments, the control value is a numerical threshold for predicting outcomes, for example good and poor outcome, or making therapy recommendations, for example adjuvant therapy in addition to surgical resection or surgical resection alone.

In some embodiments, the “control” is a predetermined value for the set of biomarkers (e.g., set of 5 to 14 biomarkers) obtained from NSCLC patients whose biomarker expression values and survival times are known. Alternatively, the “control” is a predetermined reference profile for the set of biomarkers obtained from NSCLC patients whose survival times are known. Using values from known samples allows one to develop an algorithm for classifying new patient samples into good and poor survival groups as described in the Example.

Accordingly, in one embodiment, the control is a sample from a subject known to have NSCLC and good survival outcome. In another embodiment, the control is a sample from a subject known to have NSCLC and poor survival outcome.

A person skilled in the art will appreciate that the comparison between the expression of the biomarkers in the test sample and the expression of the biomarkers in the control will depend on the control used. For example, if the control is from a subject known to have NSCLC and poor survival, and there is a difference in expression of the biomarkers between the control and test sample, then the subject can be prognosed or classified in a good survival group. If the control is from a subject known to have NSCLC and good survival, and there is a difference in expression of the biomarkers between the control and test sample, then the subject can be prognosed or classified in a poor survival group. For example, if the control is from a subject known to have NSCLC and good survival, and there is a similarity in expression of the biomarkers between the control and test sample, then the subject can be prognosed or classified in a good survival group. For example, if the control is from a subject known to have NSCLC and poor survival, and there is a similarity in expression of the biomarkers between the control and test sample, then the subject can be prognosed or classified in a poor survival group.

As used herein, a “reference value” refers to a gene-specific coefficient derived from historical expression data. The multi-gene signatures of the present disclosure comprise gene-specific reference values. In some embodiments, the multi-gene signature comprises one reference value for each gene in the signature. In some embodiments, the multi-gene signature comprises four reference values for each gene in the signature. In some embodiments, the reference values are the first four components derived from principal component analysis for each gene in the signature.

The term “differentially expressed” or “differential expression” as used herein refers to a difference in the level of expression of the biomarkers that can be assayed by measuring the level of expression of the products of the biomarkers, such as the difference in level of messenger RNA transcript expressed or proteins expressed of the biomarkers. In a preferred embodiment, the difference is statistically significant. The term “difference in the level of expression” refers to an increase or decrease in the measurable expression level of a given biomarker as measured by the amount of messenger RNA transcript and/or the amount of protein in a sample as compared with the measurable expression level of a given biomarker in a control. In one embodiment, the differential expression can be compared using the ratio of the level of expression of a given biomarker or biomarkers as compared with the expression level of the given biomarker or biomarkers of a control, wherein the ratio is not equal to 1.0. For example, an RNA or protein is differentially expressed if the ratio of the level of expression in a first sample as compared with a second sample is greater than or less than 1.0. For example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. In another embodiment the differential expression is measured using p-value. For instance, when using p-value, a biomarker is identified as being differentially expressed as between a first sample and a second sample when the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, the most preferably less than 0.001.

The term “similarity in expression” as used herein means that there is no or little difference in the level of expression of the biomarkers between the test sample and the control or reference profile. For example, similarity can refer to a fold difference compared to a control. In a preferred embodiment, there is no statistically significant difference in the level of expression of the biomarkers.

The term “most similar” in the context of a reference profile refers to a reference profile that is associated with a clinical outcome that shows the greatest number of identities and/or degree of changes with the subject profile.

The term “prognosis” as used herein refers to a clinical outcome group such as a poor survival group or a good survival group associated with a disease subtype which is reflected by a reference profile such as a biomarker reference expression profile or reflected by expression levels of the biomarkers disclosed herein. The prognosis provides an indication of disease progression and includes an indication of likelihood of death due to lung cancer. In one embodiment the clinical outcome class includes a good survival group and a poor survival group.

The term “prognosing or classifying” as used herein means predicting or identifying the clinical outcome group that a subject belongs to according to the subject's similarity to a reference profile or biomarker expression level associated with the prognosis. For example, prognosing or classifying comprises a method or process of determining whether an individual with NSCLC has a good or poor survival outcome, or grouping an individual with NSCLC into a good survival group or a poor survival group.

The term “good survival” as used herein refers to an increased chance of survival as compared to patients in the “poor survival” group. For example, the biomarkers of the application can prognose or classify patients into a “good survival group”. These patients are at a lower risk of death after surgery.

The term “poor survival” as used herein refers to an increased risk of death as compared to patients in the “good survival” group. For example, biomarkers or genes of the application can prognose or classify patients into a “poor survival group”. These patients are at greater risk of death from surgery.

Accordingly, in one embodiment, the biomarker reference expression profile comprises a poor survival group. In another embodiment, the biomarker reference expression profile comprises a good survival group.

The term “subject” as used herein refers to any member of the animal kingdom, preferably a human being that has NSCLC or that is suspected of having NSCLC.

NSCLC patients are classified into stages, which are conventionally used to determine therapy. Staging classification testing may include any or all of history, physical examination, routine laboratory evaluations, chest x-rays, and chest computed tomography scans or positron emission tomography scans with infusion of contrast materials. For example, stage I includes cancer in the lung, but has not spread to adjacent lymph nodes or outside the chest. Stage I is divided into two categories based on the size of the tumor (IA and IB). Stage II includes cancer located in the lung and proximal lymph nodes. Stage II is divided into 2 categories based on the size of tumor and nodal status (IIA and IIB). Stage III includes cancer located in the lung and the lymph nodes. Stage III is divided into 2 categories based on the size of tumor and nodal status (IIIA and IIIB). Stage IV includes cancer that has metastasized to distant locations. The term “early stage NSCLC” includes patients with Stage I to IIIA NSCLC. These patients are treated primarily by complete surgical resection.

In an aspect, a multi-gene signature is prognostic of patient outcome and/or response to adjuvant chemotherapy. In some embodiments, a minimal signature for 5 to 13 genes is provided. In one embodiment, the signature comprises reference values for each of the 5 to 13 genes listed in Table 4A. In some embodiments, the 5 to 13-gene signature is associated with the early stages of NSCLC. Accordingly, in connection with any aspect or embodiment of the invention described herein, the subject may have stage I or stage II NSCLC. In some embodiments, a 5 to 13-gene signature is prognostic of patient outcome and/or response to adjuvant chemotherapy.

In some embodiments, the multi-gene signature comprises four coefficients, or reference values, for each gene in the signature. In one embodiment, the four coefficients are the first four principal components derived from principal component analysis described in Example 1 below. In one embodiment, the 5 to 13-gene signature comprises the principal component values listed in Table 10 below.

The term “test sample” as used herein refers to any cancer-affected fluid, cell or tissue sample from a subject which can be assayed for biomarker expression products and/or a reference expression profile, e.g. genes differentially expressed in subjects with NSCLC according to survival outcome. In connection with any aspect or embodiment of the invention described herein, the test sample may be a fresh frozen or a formalin-fixed paraffin-embedded (FFPE) tumor tissue sample. As disclosed herein, such tumor specimens can be used to provide accurate gene expression profiles for the purpose of classifying samples. Further, using such samples, minimal gene sets of 5 to 14 genes may be employed. RNA may be isolated from tissues using techniques known in the art. RNA Methodologies, A laboratory guide for isolation and characterization, 2nd edition, 1998, Robert E. Farrell, Jr., Ed., Academic Press. For example, RNA may be isolated from frozen tissue samples by homogenization in guanidinium isothiocyanate and acid phenol-chloroform extraction. Commercial kits are available for isolating RNA, including for use with FFPE specimens.

The phrase “determining the expression of biomarkers” as used herein refers to determining or quantifying RNA or proteins expressed by the biomarkers. The term “RNA” includes mRNA transcripts, and/or specific spliced variants of mRNA. The terms “RNA product of the biomarker,” “biomarker RNA,” or “target RNA” as used herein refers to RNA transcripts transcribed from the biomarkers and/or specific spliced variants. In the case of “protein”, it refers to proteins translated from the RNA transcripts transcribed from the biomarkers. The term “protein product of the biomarker” or “biomarker protein” refers to proteins translated from RNA products of the biomarkers.

A person skilled in the art will appreciate that a number of methods can be used to detect or quantify the level of RNA products of the biomarkers within a sample, including arrays, such as microarrays, RT-PCR (including quantitative RT-PCR), nuclease protection assays, multiplex assays including nanostring technology, and Northern blot analyses. Any analytical procedure capable of permitting specific and quantifiable (or semi-quantifiable) detection of the biomarkers may be used in the methods herein presented, such as the microarray and quantitative PCR, e.g. quantitative RT-PCR, methods set forth herein, and methods known to those skilled in the art.

Accordingly, in one embodiment, the biomarker expression levels are determined using arrays, optionally microarrays, RT-PCR, optionally quantitative RT-PCR, nuclease protection assays or Northern blot analyses.

In some embodiments, the biomarker expression levels are determined by using an array. cDNA microarrays consist of multiple (usually thousands) of different cDNA probes spotted (usually using a robotic spotting device) onto known locations on a solid support, such as a glass microscope slide. Microarrays for use in the methods described herein comprise a solid substrate onto which the probes are covalently or non-covalently attached. The cDNAs are typically obtained by PCR amplification of plasmid library inserts using primers complementary to the vector backbone portion of the plasmid or to the gene itself for genes where sequence is known. PCR products suitable for production of microarrays are typically between 0.5 and 2.5 kB in length. In a typical microarray experiment, RNA (either total RNA or poly A RNA) is isolated from cells or tissues of interest and is reverse transcribed to yield cDNA. Labeling is usually performed during reverse transcription by incorporating a labeled nucleotide in the reaction mixture. A microarray is then hybridized with labeled RNA, and relative expression levels calculated based on the relative concentrations of cDNA molecules that hybridized to the cDNAs represented on the microarray. Microarray analysis can be performed by commercially available equipment, following manufactuer's protocols, such as by using Affymetrix GeneChip technology, Agilent Technologies cDNA microarrays, Illumina Whole-Genome DASL array assays, or any other comparable microarray technology.

In some embodiments, probes capable of hybridizing to one or more biomarker RNAs or cDNAs are attached to the substrate at a defined location (“addressable array”). Probes can be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. In some embodiments, the probes are synthesized first and subsequently attached to the substrate. In other embodiments, the probes are synthesized on the substrate. In some embodiments, probes are synthesized on the substrate surface using techniques such as photopolymerization and photolithography.

In some embodiments, microarrays are utilized in a RNA-primed, Array-based Klenow Enzyme (“RAKE”) assay. See Nelson, P. T. et al. (2004) Nature Methods 1(2):1-7; Nelson, P. T. et al. (2006) RNA 12(2):1-5, each of which is incorporated herein by reference in its entirety. In these embodiments, total RNA is isolated from a sample. Optionally, small RNAs can be further purified from the total RNA sample. The RNA sample is then hybridized to DNA probes immobilized at the 5′-end on an addressable array. The DNA probes comprise a base sequence that is complementary to a target RNA of interest, such as one or more biomarker RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence that is identically present in one of the genes listed in Table 4A under standard hybridization conditions.

In some embodiments, the addressable array comprises DNA probes for no more than the 13 genes listed in Table 4A. In some embodiments, the addressable array comprises DNA probes for each of the 13 genes listed in Table 4A and optionally, no more than one additional gene selected from those listed in Table 3.

In some embodiments, quantitation of biomarker RNA expression levels requires assumptions to be made about the total RNA per cell and the extent of sample loss during sample preparation. In some embodiments, the addressable array comprises DNA probes for each of the 13 genes listed in Table 4A and, optionally, one, two, three, or four housekeeping genes (or “normalization genes”).

In some embodiments, expression data are pre-processed to correct for variations in sample preparation or other non-experimental variables affecting expression measurements. For example, background adjustment, quantile adjustment, and summarization may be performed on microarray data, using standard software programs such as RMAexpress v0.3, followed by centering of the data to the mean and scaling to the standard deviation.

After the sample is hybridized to the array, it is exposed to exonuclease I to digest any unhybridized probes. The Klenow fragment of DNA polymerase I is then applied along with biotinylated dATP, allowing the hybridized biomarker RNAs to act as primers for the enzyme with the DNA probe as template. The slide is then washed and a streptavidin-conjugated fluorophore is applied to detect and quantitate the spots on the array containing hybridized and Klenow-extended biomarker RNAs from the sample.

In some embodiments, the RNA sample is reverse transcribed using a biotin/poly-dA random octamer primer. The RNA template is digested and the biotin-containing cDNA is hybridized to an addressable microarray with bound probes that permit specific detection of biomarker RNAs. In typical embodiments, the microarray includes at least one probe comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, even at least 20, 21, 22, 23, or 24 contiguous nucleotides identically present in each of the genes listed in Table 4A. After hybridization of the cDNA to the microarray, the microarray is exposed to a streptavidin-bound detectable marker, such as a fluorescent dye, and the bound cDNA is detected. See Liu C. G. et al. (2008) Methods 44:22-30, which is incorporated herein by reference in its entirety.

In one embodiment, the array is a U133A chip from Affymetrix. In another embodiment, a plurality of nucleic acid probes that are complementary or hybridizable to an expression product of the genes listed in Table 4A are used on the array. In a particular embodiment, the probe target sequences are listed in Table 9. In some embodiments, the probe target sequences are selected from SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169. In one embodiment, thirteen probes are used, each probe hybridizable to a different target sequence selected from SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169. In some embodiments, a plurality of nucleic acid probes that are complementary or hybridizable to an expression product of some or all the genes listed in Table 3 are used on the array. In some embodiments, the probe target sequences are selected from those listed in Table 11. In some embodiments, the probe target sequences are selected from SEQ ID NO:1-172.

The term “nucleic acid” includes DNA and RNA and can be either double stranded or single stranded.

The term “hybridize” or “hybridizable” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. may be employed.

The term “probe” as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to an RNA product of the biomarker or a nucleic acid sequence complementary thereof. The length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length.

In some embodiments, compositions are provided that comprise at least one biomarker or target RNA-specific probe. The term “target RNA-specific probe” encompasses probes that have a region of contiguous nucleotides having a sequence that is either (i) identically present in one of the genes listed in Tables 3 or 4A, or (ii) complementary to the sequence of a region of contiguous nucleotides found in one of the genes listed in Tables 3 or 4A, where “region” can comprise the full length sequence of any one of the genes listed in Tables 3 or 4A, a complementary sequence of the full length sequence of any one of the genes listed in Tables 3 or 4A, or a subsequence thereof.

In some embodiments, target RNA-specific probes consist of deoxyribonucleotides. In other embodiments, target RNA-specific probes consist of both deoxyribonucleotides and nucleotide analogs. In some embodiments, biomarker RNA-specific probes comprise at least one nucleotide analog which increases the hybridization binding energy. In some embodiments, a target RNA-specific probe in the compositions described herein binds to one biomarker RNA in the sample.

In some embodiments, more than one probe specific for a single biomarker RNA is present in the compositions, the probes capable of binding to overlapping or spatially separated regions of the biomarker RNA.

It will be understood that in some embodiments in which the compositions described herein are designed to hybridize to cDNAs reverse transcribed from biomarker RNAs, the composition comprises at least one target RNA-specific probe comprising a sequence that is identically present in a biomarker RNA (or a subsequence thereof).

In some embodiments, a biomarker RNA is capable of specifically hybridizing to at least one probe comprising a base sequence that is identically present in one of the genes listed in Table 4A. In some embodiments, a biomarker RNA is capable of specifically hybridizing to at least one nucleic acid probe comprising a sequence that is identically present in one of the genes listed in Table 3. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe, and comprises a sequence that is identical to a sequence selected from SEQ ID NO:1-172, or a sequence listed in Table 11. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe, and comprises a sequence that is identical to a sequence listed in Table 9. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe, and comprises a sequence that is identical to a sequence selected from SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169. In some embodiments, a biomarker RNA is capable of specifically hybridizing to at least one probe comprising a base sequence that is identically present in one of the genes listed in Table 4A.

In some embodiments, the composition comprises a plurality of target or biomarker RNA-specific probes each comprising a region of contiguous nucleotides comprising a base sequence that is identically present in one or more of the genes listed in Table 4A, or in a subsequence thereof. In some embodiments, the composition comprises a plurality of target or biomarker RNA-specific probes each comprising a region of contiguous nucleotides comprising a base sequence that is complementary to a sequence listed in Table 9. In some embodiments, the composition comprises a plurality of target RNA-specific probes each comprising a region of contiguous nucleotides comprising a base sequence that is complementary to a sequence selected from SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169.

As used herein, the terms “complementary” or “partially complementary” to a biomarker or target RNA (or target region thereof), and the percentage of “complementarity” of the probe sequence to that of the biomarker RNA sequence is the percentage “identity” to the reverse complement of the sequence of the biomarker RNA. In determining the degree of “complementarity” between probes used in the compositions described herein (or regions thereof) and a biomarker RNA, such as those disclosed herein, the degree of “complementarity” is expressed as the percentage identity between the sequence of the probe (or region thereof) and the reverse complement of the sequence of the biomarker RNA that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical as between the 2 sequences, dividing by the total number of contiguous nucleotides in the probe, and multiplying by 100.

In some embodiments, the microarray comprises probes comprising a region with a base sequence that is fully complementary to a target region of a biomarker RNA. In other embodiments, the microarray comprises probes comprising a region with a base sequence that comprises one or more base mismatches when compared to the sequence of the best-aligned target region of a biomarker RNA.

As noted above, a “region” of a probe or biomarker RNA, as used herein, may comprise or consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more contiguous nucleotides from a particular gene or a complementary sequence thereof. In some embodiments, the region is of the same length as the probe or the biomarker RNA. In other embodiments, the region is shorter than the length of the probe or the biomarker RNA.

In some embodiments, the microarray comprises thirteen probes each comprising a region of at least 10 contiguous nucleotides, such as at least 11 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, such as at least 18 contiguous nucleotides, such as at least 19 contiguous nucleotides, such as at least 20 contiguous nucleotides, such as at least 21 contiguous nucleotides, such as at least 22 contiguous nucleotides, such as at least 23 contiguous nucleotides, such as at least 24 contiguous nucleotides, such as at least 25 contiguous nucleotides with a base sequence that is identically present in one of the genes listed in Table 4.

In some embodiments, the microarray component comprises thirteen probes each comprising a region with a base sequence that is identically present in each of the genes listed in Table 4A. In some embodiments, the microarray comprises fourteen probes, each of which comprises a region with a base sequence that is identically present in each of the genes listed in Table 4A and, optionally, one of the genes listed in Table 3.

In another embodiment, the biomarker expression levels are determined by using quantitative RT-PCR. The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines. General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Numerous RNA isolation kits are commercially available.

In some embodiments, the primers used for quantitative RT-PCR comprise a forward and reverse primer for each of 5 to 13 genes listed in Table 4A. In one embodiment, the primers used for quantitative RT-PCR of the 5 to 13 genes are listed in Table 7 and/or Table 16. In one embodiment, primers comprising sequences identical to the sequences of SEQ ID NO: 173-198 and 203-206 are used for quantitative RT-PCR, wherein primers with sequences identical to SEQ ID NO:173-185, 203 and 204 are forward primers and primers with sequences identical to SEQ ID NO:186-198, 205 and 206 are reverse primers.

In some embodiments the analytical method used for detecting at least one biomarker RNA in the methods set forth herein includes real-time quantitative RT-PCR. See Chen, C. et al. (2005) Nucl. Acids Res. 33:e179, which is incorporated herein by reference in its entirety. Although PCR can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonuclease activity. In some embodiments, RT-PCR is done using a TaqMan® assay sold by Applied Biosystems, Inc. In a first step, total RNA is isolated from the sample. In some embodiments, the assay can be used to analyze about 10 ng of total RNA input sample, such as about 9 ng of input sample, such as about 8 ng of input sample, such as about 7 ng of input sample, such as about 6 ng of input sample, such as about 5 ng of input sample, such as about 4 ng of input sample, such as about 3 ng of input sample, such as about 2 ng of input sample, and even as little as about 1 ng of input sample containing RNA. In some embodiments, RT-PCR is done using a probe based on the Locked Nucleic Acid technology sold as Universal Probe Library (UPL) by Hoffman Laroche.

The TaqMan® assay utilizes a stem-loop primer that is specifically complementary to the 3′-end of a biomarker RNA. The step of hybridizing the stem-loop primer to the biomarker RNA is followed by reverse transcription of the biomarker RNA template, resulting in extension of the 3′ end of the primer. The result of the reverse transcription step is a chimeric (DNA) amplicon with the step-loop primer sequence at the 5′ end of the amplicon and the cDNA of the biomarker RNA at the 3′ end. Quantitation of the biomarker RNA is achieved by RT-PCR using a universal reverse primer comprising a sequence that is complementary to a sequence at the 5′ end of all stem-loop biomarker RNA primers, a biomarker RNA-specific forward primer, and a biomarker RNA sequence-specific TaqMan® probe.

The assay uses fluorescence resonance energy transfer (“FRET”) to detect and quantitate the synthesized PCR product. Typically, the TaqMan® probe comprises a fluorescent dye molecule coupled to the 5′-end and a quencher molecule coupled to the 3′-end, such that the dye and the quencher are in close proximity, allowing the quencher to suppress the fluorescence signal of the dye via FRET. When the polymerase replicates the chimeric amplicon template to which the TaqMan® probe is bound, the 5′-nuclease of the polymerase cleaves the probe, decoupling the dye and the quencher so that FRET is abolished and a fluorescence signal is generated. Fluorescence increases with each RT-PCR cycle proportionally to the amount of probe that is cleaved.

Exemplary probes for use with the five to thirteen genes, in connection with preparing gene expression profiles and classifying samples, are disclosed in Table 16. Exemplary probes for additional genes as may be desired, such as one or more additional genes from Table 3, are commercially available or may be prepared according to conventional methods.

In some embodiments, quantitation of the results of RT-PCR assays is done by constructing a standard curve from a nucleic acid of known concentration and then extrapolating quantitative information for biomarker RNAs of unknown concentration. In some embodiments, the nucleic acid used for generating a standard curve is an RNA of known concentration. In some embodiments, the nucleic acid used for generating a standard curve is a purified double-stranded plasmid DNA or a single-stranded DNA generated in vitro.

In some embodiments, where the amplification efficiencies of the biomarker nucleic acids and the endogenous reference are approximately equal, quantitation is accomplished by the comparative C_(t) (cycle threshold, e.g., the number of PCR cycles required for the fluorescence signal to rise above background) method. C_(t) values are inversely proportional to the amount of nucleic acid target in a sample. In some embodiments, C_(t) values of the target RNA of interest can be compared with a control or calibrator, such as RNA from normal tissue. In some embodiments, the C_(t) values of the calibrator and the target RNA samples of interest are normalized to an appropriate endogenous housekeeping gene (see above).

In addition to the TaqMan® assays, other RT-PCR chemistries useful for detecting and quantitating PCR products in the methods presented herein include, but are not limited to, UPL probes, Molecular Beacons, Scorpion probes and SYBR Green detection.

In some embodiments, Molecular Beacons can be used to detect and quantitate PCR products. Like TaqMan® probes, Molecular Beacons use FRET to detect and quantitate a PCR product via a probe comprising a fluorescent dye and a quencher attached at the ends of the probe. Unlike TaqMan® probes, Molecular Beacons remain intact during the PCR cycles. Molecular Beacon probes form a stem-loop structure when free in solution, thereby allowing the dye and quencher to be in close enough proximity to cause fluorescence quenching. When the Molecular Beacon hybridizes to a target, the stem-loop structure is abolished so that the dye and the quencher become separated in space and the dye fluoresces. Molecular Beacons are available, e.g., from Gene Link™ (see http://www.genelink.com/newsite/products/mbintro.asp).

In some embodiments, Scorpion probes can be used as both sequence-specific primers and for PCR product detection and quantitation. Like Molecular Beacons, Scorpion probes form a stem-loop structure when not hybridized to a target nucleic acid. However, unlike Molecular Beacons, a Scorpion probe achieves both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5′-end of the Scorpion probe, and a quencher is attached to the 3′-end. The 3′ portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is linked to the 5′-end of the probe by a non-amplifiable moiety. After the Scorpion primer is extended, the target-specific sequence of the probe binds to its complement within the extended amplicon, thus opening up the stem-loop structure and allowing the dye on the 5′-end to fluoresce and generate a signal. Scorpion probes are available from, e.g, Premier Biosoft International (see http://www.premierbiosoft.com/tech_notes/Scorpion.html).

In some embodiments, RT-PCR detection is performed specifically to detect and quantify the expression of a single biomarker RNA. The biomarker RNA, in typical embodiments, is selected from a biomarker RNA capable of specifically hybridizing to a nucleic acid comprising a sequence that is identically present in one of the genes set forth in Table 4A. In some embodiments, the biomarker RNA specifically hybridizes to a nucleic acid comprising a sequence that is identically present in at least one of the genes in Table 3.

In various other embodiments, RT-PCR detection is utilized to detect, in a single multiplex reaction, each of 5 to 14 (e.g., 13) biomarker RNAs. The biomarker RNAs, in some embodiments, are capable of specifically hybridizing to a nucleic acid comprising a sequence that is identically present in one of the thirteen genes listed in Table 4A.

In some multiplex embodiments, a plurality of probes, such as TaqMan probes, each specific for a different RNA target, is used. In typical embodiments, each target RNA-specific probe is spectrally distinguishable from the other probes used in the same multiplex reaction.

In some embodiments, quantitation of RT-PCR products is accomplished using a dye that binds to double-stranded DNA products, such as SYBR Green. In some embodiments, the assay is the QuantiTect SYBR Green PCR assay from Qiagen. In this assay, total RNA is first isolated from a sample. Total RNA is subsequently poly-adenylated at the 3′-end and reverse transcribed using a universal primer with poly-dT at the 5′-end. In some embodiments, a single reverse transcription reaction is sufficient to assay multiple biomarker RNAs. RT-PCR is then accomplished using biomarker RNA-specific primers and an miScript Universal Primer, which comprises a poly-dT sequence at the 5′-end. SYBR Green dye binds non-specifically to double-stranded DNA and upon excitation, emits light. In some embodiments, buffer conditions that promote highly-specific annealing of primers to the PCR template (e.g., available in the QuantiTect SYBR Green PCR Kit from Qiagen) can be used to avoid the formation of non-specific DNA duplexes and primer dimers that will bind SYBR Green and negatively affect quantitation. Thus, as PCR product accumulates, the signal from SYBR green increases, allowing quantitation of specific products.

RT-PCR is performed using any RT-PCR instrumentation available in the art. Typically, instrumentation used in real-time RT-PCR data collection and analysis comprises a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

In some embodiments, the method of detectably quantifying one or more biomarker RNAs includes the steps of: (a) isolating total RNA; (b) reverse transcribing a biomarker RNA to produce a cDNA that is complementary to the biomarker RNA; (c) amplifying the cDNA from step (b); and (d) detecting the amount of a biomarker RNA with RT-PCR.

As described above, in some embodiments, the RT-PCR detection is performed using a FRET probe, which includes, but is not limited to, a TaqMan® probe, a Molecular beacon probe and a Scorpion probe. In some embodiments, the RT-PCR detection and quantification is performed with a TaqMan® probe, i.e., a linear probe that typically has a fluorescent dye covalently bound at one end of the DNA and a quencher molecule covalently bound at the other end of the DNA. The FRET probe comprises a base sequence that is complementary to a region of the cDNA such that, when the FRET probe is hybridized to the cDNA, the dye fluorescence is quenched, and when the probe is digested during amplification of the cDNA, the dye is released from the probe and produces a fluorescence signal. In such embodiments, the amount of biomarker RNA in the sample is proportional to the amount of fluorescence measured during cDNA amplification.

The TaqMan® probe typically comprises a region of contiguous nucleotides comprising a base sequence that is complementary to a region of a biomarker RNA or its complementary cDNA that is reverse transcribed from the biomarker RNA template (i.e., the sequence of the probe region is complementary to or identically present in the biomarker RNA to be detected) such that the probe is specifically hybridizable to the resulting PCR amplicon. In some embodiments, the probe comprises a region of at least 6 contiguous nucleotides having a base sequence that is fully complementary to or identically present in a region of a cDNA that has been reverse transcribed from a biomarker RNA template, such as comprising a region of at least 8 contiguous nucleotides, or comprising a region of at least 10 contiguous nucleotides, or comprising a region of at least 12 contiguous nucleotides, or comprising a region of at least 14 contiguous nucleotides, or even comprising a region of at least 16 contiguous nucleotides having a base sequence that is complementary to or identically present in a region of a cDNA reverse transcribed from a biomarker RNA to be detected.

Preferably, the region of the cDNA that has a sequence that is complementary to the TaqMan® probe sequence is at or near the center of the cDNA molecule. In some embodiments, there are independently at least 2 nucleotides, such as at least 3 nucleotides, such as at least 4 nucleotides, such as at least 5 nucleotides of the cDNA at the 5′-end and at the 3′-end of the region of complementarity.

In typical embodiments, all biomarker RNAs are detected in a single multiplex reaction. In these embodiments, each TaqMan® probe that is targeted to a unique cDNA is spectrally distinguishable when released from the probe. Thus, each biomarker RNA is detected by a unique fluorescence signal.

In some embodiments, expression levels may be represented by gene transcript numbers per nanogram of cDNA. To control for variability in cDNA quantity, integrity and the overall transcriptional efficiency of individual primers, RT-PCR data can be subjected to standardization and normalization against one or more housekeeping genes as has been previously described. See e.g., Rubie et al., Mol. Cell. Probes 19(2):101-9 (2005).

Appropriate genes for normalization in the methods described herein include those as to which the quantity of the product does not vary between between different cell types, cell lines or under different growth and sample preparation conditions. In some embodiments, endogenous housekeeping genes useful as normalization controls in the methods described herein include, but are not limited to, ACTB, BAT1, B2M, EDS, IP08, TBP, PP1A, GUSB, U6 snRNA, RNU44, RNU 48, and U47. In typical embodiments, at least one endogenous housekeeping gene for use in normalizing the measured quantity of RNA is selected from ACTB, BAT1, B2M, EDS, TBP, U6 snRNA, U6 snRNA, RNU44, RNU 48, and U47. For example, the methods and kits of the invention may employ 2, 3, or 4 normalization genes selected from IP08, BAT1, TBP, PP1A, and GUSB. In certain embodiments, the tissue sample is frozen, and the normalization genes include 2, 3, or 4 of IP08, BAT1, TBP, and PP1A. In other embodiments, the sample is an FFPE sample, and the normalization genes include 2, 3, or 4 of BAT1, TBP, PP1A, and GUSB. In some embodiments, one housekeeping gene is used for normalization. In some embodiments, two, three, four or more housekeeping genes are used for normalization.

In some embodiments, labels that can be used on the FRET probes include colorimetric and fluorescent labels such as Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

Specific examples of dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2′,4′,5′,7′-Tetrabromosulfonefluorescein, and TET.

Specific examples of fluorescently labeled ribonucleotides useful in the preparation of RT-PCR probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

Examples of fluorescently labeled deoxyribonucleotides useful in the preparation of RT-PCR probes for use in the methods described herein include Dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially available and can be purchased from, e.g., Invitrogen.

In some embodiments, dyes and other moieties, such as quenchers, are introduced into nucleic acids used in the methods described herein, such as FRET probes, via modified nucleotides. A “modified nucleotide” refers to a nucleotide that has been chemically modified, but still functions as a nucleotide. In some embodiments, the modified nucleotide has a chemical moiety, such as a dye or quencher, covalently attached, and can be introduced into an oligonucleotide, for example, by way of solid phase synthesis of the oligonucleotide. In other embodiments, the modified nucleotide includes one or more reactive groups that can react with a dye or quencher before, during, or after incorporation of the modified nucleotide into the nucleic acid. In specific embodiments, the modified nucleotide is an amine-modified nucleotide, i.e., a nucleotide that has been modified to have a reactive amine group. In some embodiments, the modified nucleotide comprises a modified base moiety, such as uridine, adenosine, guanosine, and/or cytosine. In specific embodiments, the amine-modified nucleotide is selected from 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Many amine modified nucleotides are commercially available from, e.g., Applied Biosystems, Sigma, Jena Bioscience and Tri Link.

In some embodiments, the methods of detecting at least one biomarker RNA described herein employ one or more modified oligonucleotides, such as oligonucleotides comprising one or more affinity-enhancing nucleotides. Modified oligonucleotides useful in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity of an oligonucleotide for its target nucleic acid as compared to oligonucleotides that contain only deoxyribonucleotides, and allows for the use of shorter oligonucleotides or for shorter regions of complementarity between the oligonucleotide and the target nucleic acid.

In some embodiments, affinity-enhancing nucleotides include nucleotides comprising one or more base modifications, sugar modifications and/or backbone modifications.

In some embodiments, modified bases for use in affinity-enhancing nucleotides include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine and hypoxanthine.

In some embodiments, affinity-enhancing modifications include nucleotides having modified sugars such as 2′-substituted sugars, such as 2′-O-alkyl-ribose sugars, 2′-amino-deoxyribose sugars, 2′-fluoro-deoxyribose sugars, 2′-fluoro-arabinose sugars, and 2′-O-methoxyethyl-ribose (2′MOE) sugars. In some embodiments, modified sugars are arabinose sugars, or d-arabino-hexitol sugars.

In some embodiments, affinity-enhancing modifications include backbone modifications such as the use of peptide nucleic acids (e.g., an oligomer including nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.

In some embodiments, the oligomer includes at least one affinity-enhancing nucleotide that has a modified base, at least nucleotide (which may be the same nucleotide) that has a modified sugar, and at least one internucleotide linkage that is non-naturally occurring.

In some embodiments, the affinity-enhancing nucleotide contains a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In some embodiments, an oligonucleotide for use in the methods described herein comprises one or more nucleotides having an LNA sugar. In some embodiments, the oligonucleotide contains one or more regions consisting of nucleotides with LNA sugars. In other embodiments, the oligonucleotide contains nucleotides with LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11):1138-1142.

The term “primer” as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art. In one embodiment, primer sets for 5 to 13 genes whose expression levels are determined, are selected from Table 7 and/or Table 16.

In addition, a person skilled in the art will appreciate that a number of methods can be used to determine the amount of a protein product of the biomarker of the invention, including immunoassays such as Western blots, ELISA, and immunoprecipitation followed by SDS-PAGE and immunocytochemistry.

Accordingly, in another embodiment, an antibody is used to detect the polypeptide products of the biomarkers listed in Table 4A. In another embodiment, the sample comprises a tissue sample. In a further embodiment, the tissue sample is suitable for immunohistochemistry.

The term “antibody” as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term “antibody fragment” as used herein is intended to include Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab′)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

Conventional techniques of molecular biology, microbiology and recombinant DNA techniques are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes & S. J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal, 1984); and a series, Methods in Enzymology (Academic Press, Inc.); Short Protocols In Molecular Biology, (Ausubel et al., ed., 1995).

For example, antibodies having specificity for a specific protein, such as the protein product of a biomarker, may be prepared by conventional methods. A mammal, (e.g. a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g. the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.

In some embodiments, recombinant antibodies are provided that specifically bind protein products of the genes listed in Table 4, and optionally the expression product(s) of one gene selected from among those listed in Table 3. Recombinant antibodies include, but are not limited to, chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, single-chain antibodies and multi-specific antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody (mAb) and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety). Single-chain antibodies have an antigen binding site and consist of single polypeptides. They can be produced by techniques known in the art, for example using methods described in Ladner et al, U.S. Pat. No. 4,946,778 (which is incorporated herein by reference in its entirety); Bird et al., (1988) Science 242:423-426; Whitlow et al., (1991) Methods in Enzymology 2:1-9; Whitlow et al., (1991) Methods in Enzymology 2:97-105; and Huston et al., (1991) Methods in Enzymology Molecular Design and Modeling: Concepts and Applications 203:46-88. Multi-specific antibodies are antibody molecules having at least two antigen-binding sites that specifically bind different antigens. Such molecules can be produced by techniques known in the art, for example using methods described in Segal, U.S. Pat. No. 4,676,980 (the disclosure of which is incorporated herein by reference in its entirety); Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Whitlow et al., (1994) Protein Eng 7:1017-1026 and U.S. Pat. No. 6,121,424.

Monoclonal antibodies directed against any of the expression products of the genes listed in Table 4A and can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide(s) of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.

Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

In some embodiments, humanized antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide corresponding to a protein product. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, Calif.), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Antibodies may be isolated after production (e.g., from the blood or serum of the subject) or synthesis and further purified by well-known techniques. For example, IgG antibodies can be purified using protein A chromatography. Antibodies specific for a protein can be selected or (e.g., partially purified) or purified by, e.g., affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) expression product may be produced, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the protein products of the genes listed in Tables 3 and 4A from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition it is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the protein products of the genes listed in Tables 3 and 4A, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein.

In some embodiments, substantially purified antibodies may specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain or cytoplasmic membrane of a protein product of one of the genes listed in Table 4A. In an embodiment, substantially purified antibodies specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of a protein product of one of the genes listed in Tables 3 and 4A.

In some embodiments, antibodies directed against a protein product of one of the genes listed in Tables 3 and 4A can be used to detect the protein products or fragment thereof (e.g., in a cellular lysate or cell supernatant) in order to evaluate the level and pattern of expression of the protein. Detection can be facilitated by the use of an antibody derivative, which comprises an antibody coupled to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

A variety of techniques can be employed to measure expression levels of each of the thirteen genes in Table 4 as well as the genes in Table 3, given a sample that contains protein products that bind to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining protein expression levels of the thirteen products of the genes listed in Tables 4 and optionally additional products of one gene selected from those listed in Table 3.

In one embodiment, antibodies, or antibody fragments or derivatives, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In some embodiments, either the antibodies or proteins are immobilized on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present disclosure. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

Immunohistochemistry methods are also suitable for detecting the expression levels of the prognostic markers. In some embodiments, antibodies or antisera, including polyclonal antisera, and monoclonal antibodies specific for each marker may be used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. These assays and their quantitation against purified, labeled standards are well known in the art (Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

Numerous labels are available which can be generally grouped into the following categories:

-   -   (a) Radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The         antibody variant can be labeled with the radioisotope using the         techniques described in Current Protocols in Immunology, vol         1-2, Coligen et al., Ed., Wiley-Interscience, New York,         Pubs. (1991) for example and radioactivity can be measured using         scintillation counting.     -   (b) Fluorescent labels such as rare earth chelates (europium         chelates) or fluorescein and its derivatives, rhodamine and its         derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are         available. The fluorescent labels can be conjugated to the         antibody variant using the techniques disclosed in Current         Protocols in Immunology, supra, for example. Fluorescence can be         quantified using a fluorimeter.     -   (c) Various enzyme-substrate labels are available and U.S. Pat.         Nos. 4,275,149, 4,318,980 provides a review of some of these.         The enzyme generally catalyzes a chemical alteration of the         chromogenic substrate which can be measured using various         techniques. For example, the enzyme may catalyze a color change         in a substrate, which can be measured spectrophotometrically.         Alternatively, the enzyme may alter the fluorescence or         chemiluminescence of the substrate. Techniques for quantifying a         change in fluorescence are described above. The chemiluminescent         substrate becomes electronically excited by a chemical reaction         and may then emit light which can be measured (using a         chemiluminometer, for example) or donates energy to a         fluorescent acceptor. Examples of enzymatic labels include         luciferases (e.g., firefly luciferase and bacterial luciferase;         U.S. Pat. No. 4,737,456), luciferin,         2,3-dihydrophthalazinediones, malate dehydrogenase, urease,         peroxidase such as horseradish peroxidase (HRPO), alkaline         phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,         saccharide oxidases (e.g., glucose oxidase, galactose oxidase,         and glucose-6-phosphate dehydrogenase), heterocyclic oxidases         (such as uricase and xanthine oxidase), lactoperoxidase,         microperoxidase, and the like. Techniques for conjugating         enzymes to antibodies are described in O'Sullivan et al.,         Methods for the Preparation of Enzyme-Antibody Conjugates for         Use in Enzyme Immunoassay, in Methods in Enzyme. (Ed. J. Langone         & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

In some embodiments, a detection label is indirectly conjugated with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody). In some embodiments, the antibody need not be labeled, and the presence thereof can be detected using a labeled antibody, which binds to the antibody.

The gene signatures described herein can be used to select treatment for NCSLC patients. As explained herein, the biomarkers can classify patients with NSCLC into a poor survival group or a good survival group and into groups that might benefit from adjuvant chemotherapy or not.

Accordingly, in one embodiment, the application provides a method of selecting a therapy for a subject with NSCLC, comprising the steps:

(a) classifying the subject with NSCLC into a poor survival group or a good survival group according to the methods described herein; and

(b) selecting adjuvant chemotherapy for the subject classified as being in the poor survival group or no adjuvant chemotherapy for the subject classified as being in the good survival group.

In another embodiment, the application provides a method of selecting a therapy for a subject with NSCLC, comprising the steps:

(a) determining the expression of from 5 to thirteen biomarkers in a test sample from the subject, wherein the five to thirteen biomarkers correspond to the genes in Table 4;

(b) comparing the expression of the five to thirteen biomarkers in the test sample with the five to thirteen biomarkers in a control sample;

(c) classifying the subject in a poor survival group or a good survival group, wherein a difference or a similarity in the expression of the five to thirteen biomarkers between the control sample and the test sample is used to classify the subject into a poor survival group or a good survival group; and

(d) selecting adjuvant chemotherapy if the subject is classified in the poor survival group and selecting no adjuvant chemotherapy if the subject is classified in the good survival group.

The term “adjuvant chemotherapy” as used herein means treatment of cancer with chemotherapeutic agents after surgery where all detectable disease has been removed, but where there still remains a risk of small amounts of remaining cancer. Typical chemotherapeutic agents include cisplatin, carboplatin, vinorelbine, gemcitabine, doccetaxel, paclitaxel and navelbine.

In another aspect, the application provides compositions useful in detecting changes in the expression levels of the 13 genes listed in Table 4A. Accordingly in one embodiment, the application provides a composition comprising a plurality of isolated nucleic acid sequences wherein each isolated nucleic acid sequence hybridizes to:

(a) a RNA product of one of the 13 genes listed in Table 4; and/or

(b) a nucleic acid complementary to a),

wherein the composition is used to measure the level of RNA expression of the 13 genes. In a particular embodiment, the plurality of isolated nucleic acid sequences comprise isolated nucleic acids hybridizable to the 13 probe target sequences as set out in Table 9. In one embodiment, the plurality of isolated nucleic acid sequences comprise isolated nucleic acids hybridizable to SEQ ID NO 4, 11-15, 22, 26, 35, 78, 130, 133, and 169.

In another embodiment, the application provides a composition comprising 13 forward and 13 reverse primers for amplifying a region of each gene listed in Table 4A. In a particular embodiment, the 26 primers are as set out in Table 7. An additional primer set is described in Table 16. Any combination of the primer sets listed in Table 7 and Table 16 can be used to amplify the genes listed in Table 4A. In one embodiment, the 26 primers each comprise a sequence that is identical to the sequence of one of SEQ ID NO: 173-198 and 203-206. In certain embodiments, L1CAM cDNA is amplified using the forward and revserse primers disclosed in Table 16. In this or another embodiment, MDM2 cDNA is amplified using forward and reverse primers disclosed in Table 16.

In a further aspect, the application also provides an array that is useful in detecting the expression levels of the 13 genes set out in Table 4A (or a subset thereof including at least 5 genes from Table 4A). Accordingly, in one embodiment, the application provides an array comprising for each gene shown in Table 4A (or the subset) one or more nucleic acid probes complementary and hybridizable to an expression product of the gene. In a particular embodiment, the array comprises the nucleic acid probes hybridizable to the probe target sequences listed in Table 9. In one embodiment, the array comprises the nucleic acid probes hybridizable to sequences identical to each of SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169.

In yet another aspect, the application also provides for kits used to prognose or classify a subject with NSCLC into a good survival group or a poor survival group or to select a therapy for a subject with NSCLC that includes detection agents that can detect the expression products of the biomarkers. Accordingly, in one embodiment, the application provides a kit to prognose or classify a subject with early stage NSCLC comprising detection agents that can detect the expression products of from 5 to 14 biomarkers, wherein the 5 to 14 biomarkers comprise from 5 to 13 genes in Table 4A. The set of up to 14 biomarkers may further comprise genes from Table 3.

In one embodiment, the application provides a kit to select a therapy for a subject with NSCLC, comprising detection agents that can detect the expression products of from 5 to 14 biomarkers, wherein the 5 to 14 biomarkers comprise 5 to 13 genes in Table 4A, and optionally genes from Table 3.

For example, the kit may comprise a primer set for amplifying a target sequence in each of from 5 to 14 genes from Table 3, at least 5 of which are from Table 4 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 genes from Table 4A). Exemplary target sequences are shown in Table 11. In certain embodiments, the primer set contains primer pairs (forward and reverse primers) for amplifying 5 to 13 genes from Table 4A. In certain embodiments, the kit further comprises a primer set for amplifying at least one normalization gene, such as one or more normalization genes described herein. Additionally, the kit may comprise at least one probe for detecting each target sequence, including in connection with the detection platforms described herein (e.g., TaqMan™).

The materials and methods of the present disclosure are ideally suited for preparation of kits produced in accordance with well known procedures. In some embodiments, kits comprise agents (like the polynucleotides and/or antibodies described herein as non-limiting examples) for the detection of expression of the disclosed sequences, such as for example, SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169, the target sequences listed in Table 9, or the target sequences listed in Table 11. Kits, may comprise containers, each with one or more of the various reagents (sometimes in concentrated form), for example, pre-fabricated microarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more primer complexes (e.g., appropriate length poly(T) or random primers linked to a promoter reactive with the RNA polymerase). A set of instructions will also typically be included.

In some embodiments, a kit may comprise a plurality of reagents, each of which is capable of binding specifically with a target nucleic acid or protein. Suitable reagents for binding with a target protein include antibodies, antibody derivatives, antibody fragments, and the like. Suitable reagents for binding with a target nucleic acid (e.g. a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleic acids. For example, nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.

In some embodiments, kits may comprise additional components useful for detecting gene expression levels. By way of example, kits may comprise fluids (e.g. SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample compartments, a material which provides instruction for detecting expression levels, and the like.

In some embodiments, kits for use in the RT-PCR methods described herein comprise one or more target RNA-specific FRET probes and one or more primers for reverse transcription of target RNAs or amplification of cDNA reverse transcribed therefrom.

In some embodiments, one or more of the primers is “linear”. A “linear” primer refers to an oligonucleotide that is a single stranded molecule, and typically does not comprise a short region of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to another region within the same oligonucleotide such that the primer forms an internal duplex. In some embodiments, the primers for use in reverse transcription comprise a region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more contiguous nucleotides at the 3′-end that has a base sequence that is complementary to region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more contiguous nucleotides at the 5′-end of a target RNA.

In some embodiments, the kit further comprises one or more pairs of linear primers (a “forward primer” and a “reverse primer”) for amplification of a cDNA reverse transcribed from a target RNA. Accordingly, in some embodiments, the forward primer comprises a region of at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 contiguous nucleotides having a base sequence that is complementary to the base sequence of a region of at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 contiguous nucleotides at the 5′-end of a target RNA. Furthermore, in some embodiments, the reverse primer comprises a region of at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 contiguous nucleotides having a base sequence that is complementary to the base sequence of a region of at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 contiguous nucleotides at the 3′-end of a target RNA.

In some embodiments, the kit comprises at least a first set of primers for amplification of a cDNA that is reverse transcribed from a target RNA capable of specifically hybridizing to a nucleic acid comprising a sequence identically present in one of the genes listed in Table 4A. In some embodiments, the kit comprises at least thirteen sets of primers, each of which is for amplification of a different target RNA capable of specifically hybridizing to a nucleic acid comprising a sequence identically present in a different gene listed in Table 4A. In one embodiment, the kit comprises thirteen forward and thirteen reverse primers described in Table 7, comprising sequences identical to SEQ ID NOs 173-198. In some embodiments, the kit comprises at least one set of primers that is capable of amplifying more than one cDNA reverse transcribed from a target RNA in a sample.

In some embodiments, probes and/or primers for use in the compositions described herein comprise deoxyribonucleotides. In some embodiments, probes and/or primers for use in the compositions described herein comprise deoxyribonucleotides and one or more nucleotide analogs, such as LNA analogs or other duplex-stabilizing nucleotide analogs described above. In some embodiments, probes and/or primers for use in the compositions described herein comprise all nucleotide analogs. In some embodiments, the probes and/or primers comprise one or more duplex-stabilizing nucleotide analogs, such as LNA analogs, in the region of complementarity.

In some embodiments, the compositions described herein also comprise probes, and in the case of RT-PCR, primers, that are specific to one or more housekeeping genes for use in normalizing the quantities of target RNAs. Such probes (and primers) include those that are specific for one or more products of housekeeping genes selected from ACTB, BAT1, B2M, EDS, IP08, TBP, PPA1, GUSB, U6 snRNA, RNU44, RNU 48, and U47.

In some embodiments, the kits for use in real time RT-PCR methods described herein further comprise reagents for use in the reverse transcription and amplification reactions. In some embodiments, the kits comprise enzymes such as reverse transcriptase, and a heat stable DNA polymerase, such as Taq polymerase. In some embodiments, the kits further comprise deoxyribonucleotide triphosphates (dNTP) for use in reverse transcription and amplification. In further embodiments, the kits comprise buffers optimized for specific hybridization of the probes and primers.

In some embodiments, kits are provided containing antibodies to each of the protein products of the genes listed in Table 4A, conjugated to a detectable substance, and instructions for use. Kits may comprise an antibody, an antibody derivative, or an antibody fragment, which binds specifically with a marker protein, or a fragment of the protein. Such kits may also comprise a plurality of antibodies, antibody derivatives, or antibody fragments wherein the plurality of such antibody agents binds specifically with a marker protein, or a fragment of the protein.

In some embodiments, kits may comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such kits can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail herein for nucleic acid arrays and similar methods have been developed for antibody arrays.

A person skilled in the art will appreciate that a number of detection agents can be used to determine the expression of the biomarkers. For example, to detect RNA products of the biomarkers, probes, primers, complementary nucleotide sequences or nucleotide sequences that hybridize to the RNA products can be used. To detect protein products of the biomarkers, ligands or antibodies that specifically bind to the protein products can be used.

Accordingly, in one embodiment, the detection agents are probes that hybridize to the 13 biomarkers. In a particular embodiment, the probe target sequences are as set out in Table 9. In one embodiment, the probe target sequences are identical to SEQ ID NO: 4, 11-15, 22, 26, 35, 78, 130, 133, and 169. In another embodiment, the detection agents are forward and reverse primers that amplify a region of each of the 13 genes listed in Table 4A. In a particular embodiment, the primers are as set out in Table 7 and Table 16. In one embodiment, the primers comprise one or more of the polynucleotide sequences (or one or more primer sets) of SEQ ID NO: 173-198 and 203-206.

A person skilled in the art will appreciate that the detection agents can be labeled.

The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.

The kit can also include a control or reference standard and/or instructions for use thereof. In addition, the kit can include ancillary agents such as vessels for storing or transporting the detection agents and/or buffers or stabilizers.

In some aspects, a multi-gene signature is provided for prognosis or classifying patients with lung cancer. In some embodiments, a 5 to fourteen (e.g., thirteen)-gene signature is provided, comprising reference values for each of the genes based on relative expression data from a historical data set with a known outcome, such as good or poor survival, and/or known treatment, such as adjuvant chemotherapy. In one embodiment, four reference values are provided for each of the thirteen genes listed in Table 4A. In one embodiment, the reference values for each of the thirteen genes are principal component values set forth in Table 10.

In one aspect, relative expression data from a patient are combined with the gene-specific reference values on a gene-by-gene basis for each of the thirteen, and, optionally, additional genes, to generate a test value which allows prognosis or therapy recommendation. In some embodiments, relative expression data are subjected to an algorithm that yields a single test value, or combined score, which is then compared to a control value obtained from the historical expression data for a patient or pool of patients.

In some embodiments, the control value is a numerical threshold for predicting outcomes, for example good and poor outcome, or making therapy recommendations for a subject, for example adjuvant chemotherapy in addition to surgical resection or surgical resection alone. In some embodiments, a test value or combined score greater than the control value is predictive, for example, of a poor outcome or benefit from adjuvant chemotherapy, whereas a combined score falling below the control value is predictive, for example, of a good outcome or lack of benefit from adjuvant chemotherapy for a subject.

In some embodiments, a method for prognosing or classifying a subject with NSCLC comprises:

-   -   (a) measuring expression levels of from 5 to 14 (e.g., 13)         biomarkers from Table 4 in a test sample,     -   (b) calculating a combined score or test value for the subject         from the expression levels, and,     -   (c) comparing the combined score to a control value,         Wherein a combined score greater than the control value is used         to classify a subject into a high risk or poor survival group         and a combined score lower than the control value is used to         classify a subject into a lower risk or good survival group.

In one embodiment, the combined score is calculated from relative expression data multiplied by reference values, determined from historical data, for each gene. Accordingly, the combined score may be calculated using Formula I below:

Combined score=0.557×PC1+0.328×PC2+0.43×PC3+0.335×PC4

Where PC1 is the sum of the relative expression level for each gene in a multi-gene signature multiplied by a first principal component for each gene in the multi-gene signature, PC2 is the sum of the relative expression level for each gene multiplied by a second principal component for each gene, PC3 is the sum of the relative expression level for each gene multiplied by a third principal component for each gene, and PC4 is the sum of the relative expression level for each gene multiplied by a fourth principal component for each gene. In some embodiments, the combined score is referred to as a risk score. A risk score for a subject can be calculated by applying Formula I to relative expression data from a test sample obtained from the subject.

In some embodiments, PC1 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a first principal component for each gene, respectively, as set forth in Table 10; PC2 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a second principal component for each gene, respectively, as set forth in Table 10; PC3 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a third principal component for each gene, respectively, as set forth in Table 10; and PC4 is the sum of the relative expression level for each gene provided in Table 4A multiplied by a fourth principal component for each gene, respectively, as set forth in Table 10.

In one embodiment, the control value is equal to −0.1. A subject with a risk score of more than −0.1 is classified as high risk (poor prognosis). A patient with a risk score of less than −0.1 is classified as lower risk (good prognosis). In some embodiments, adjuvant chemotherapy is recommended for a subject with a risk score of more than −0.1 and not recommended for a subject with a risk score of less than −0.1.

In some embodiments, the invention involves classifying the sample into a high risk or low risk group as described herein. For example, samples may be classified on the basis of threshold values or based upon Mean and/or Median expression levels in high risk patients versus low-risk patients. Various classification schemes are known for classifying samples between two or more classes or groups, and these include, without limitation: Principal Components Analysis, Naïve Bayes, Support Vector Machines, Nearest Neighbors, Decision Trees, Logistic, Artificial Neural Networks, Penalized Logistic Regression, and Rule-based schemes. In addition, the predictions from multiple models can be combined to generate an overall prediction. For example, a “majority rules” prediction may be generated from the outputs of a Naïve Bayes model, a Support Vector Machine model, and a Nearest Neighbor model.

Thus, a classification algorithm or “class predictor” may be constructed to classify samples. The process for preparing a suitable class predictor is reviewed in R. Simon, Diagnostic and prognostic prediction using gene expression profiles in high-dimensional microarray data, British Journal of Cancer (2003) 89, 1599-1604, which review is hereby incorporated by reference in its entirety.

In a further aspect, the application provides computer programs and computer implemented products for carrying out the methods described herein. Accordingly, in one embodiment, the application provides a computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, the computer program product comprising a computer readable storage medium having a computer mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the computer to carry out the methods described herein.

In another embodiment, the application provides a computer implemented product for predicting a prognosis or classifying a subject with NSCLC comprising:

(a) a means for receiving values corresponding to a subject expression profile in a subject sample; and

(b) a database comprising a reference expression profile associated with a prognosis, wherein the subject biomarker expression profile and the biomarker reference profile each has thirteen or fourteen values, each value representing the expression level of a biomarker, wherein each biomarker corresponds to one gene in Table 4A;

wherein the computer implemented product selects the biomarker reference expression profile most similar to the subject biomarker expression profile, to thereby predict a prognosis or classify the subject.

In yet another embodiment, the application provides a computer implemented product for determining therapy for a subject with NSCLC comprising:

(a) a means for receiving values corresponding to a subject expression profile in a subject sample; and

(b) a database comprising a reference expression profile associated with a therapy, wherein the subject biomarker expression profile and the biomarker reference profile each has thirteen or fourteen values, each value representing the expression level of a biomarker, wherein each biomarker corresponds to one gene in Table 4A;

wherein the computer implemented product selects the biomarker reference expression profile most similar to the subject biomarker expression profile, to thereby predict the therapy.

Another aspect relates to computer readable mediums such as CD-ROMs. In one embodiment, the application provides computer readable medium having stored thereon a data structure for storing a computer implemented product described herein.

In one embodiment, the data structure is capable of configuring a computer to respond to queries based on records belonging to the data structure, each of the records comprising:

(a) a value that identifies a biomarker reference expression profile of the 13 genes in Table 4A;

(b) a value that identifies the probability of a prognosis associated with the biomarker reference expression profile.

In another aspect, the application provides a computer system comprising

(a) a database including records comprising a biomarker reference expression profile of thirteen genes in Table 4A associated with a prognosis or therapy;

(b) a user interface capable of receiving a selection of gene expression levels of the 13 genes in Table 4A for use in comparing to the biomarker reference expression profile in the database; and

(c) an output that displays a prediction of prognosis or therapy according to the biomarker reference expression profile most similar to the expression levels of the thirteen genes.

In some embodiments, the application provides a computer implemented product comprising

(a) a means for receiving values corresponding to relative expression levels in a subject, of at least 13 biomarkers comprising the thirteen genes in Table 4A;

(b) an algorithm for calculating a combined scire based on the relative expression levels of the at least 13 biomarkers;

(c) an output that displays the combined score; and, optionally,

(d) an output that displays a prognosis or therapy recommendation based on the combined score.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

The following non-limiting example is illustrative of the present invention:

Example 1 Results

Table 1 compares the demographic features of 133 patients with microarray profiling to 349 without the profiling. Stage 1B patients had more representation in the observation cohort (55% vs. 42%, p=0.01), but all other factors were similarly distributed. There was no significant difference in the overall survivals of patients with or without gene profiling (FIG. 2A). For these 133 patients, adjuvant chemotherapy reduced the death rate by 20% (HR 0.80, 95% Cl 0.48-1.32, p=0.38; FIG. 5).

Prognostic Gene Expression Signature in JBR.10 Patients

Using a p>0.005 as cut-off, 172 of 19,619 probe sets were significantly associated with prognosis in 62 observation patients (FIG. 1A and Table 3). Using a method that was designed to identify the minimum expression gene set that can distinguish most patients with poor and good survival outcomes, a 15-gene prognostic signature was identified (FIG. 1A and Table 4 and 4b). This signature was able to separate the 62 non-adjuvant treated patients into 31 low-risk and 31 high-risk patients for death (HR 15.020, 95% Cl 5.12-44.04, p<0.0001; FIG. 2B). Furthermore, stratified analysis showed that the signature was also highly prognostic in 34 stage IB patients (HR 13.32, 95% Cl 2.86-62.11, p<0.0001, FIG. 2C) and 28 stage II patients (HR 13.47, 95% Cl 3.0-60.43, p<0.0001, FIG. 2D). Multivariate analysis adjusting for tumor stage, age, gender and histology showed that the prognostic signature was an independent prognostic marker (HR 18.0, 95% Cl 5.8-56.1; p<0.0001, Table 2). This did not differ following additional adjustment for surgical procedure and tumor size. Further analysis shows that 2 genes, MLANA and MYT1L, have no significant effect on the risk score (FIG. 7). Consequently, a minimal 13-gene signature is defined.

Validation of General Applicability of Prognostic Signature (Summary)

Applying the risk score algorithm (equation) established from the 62 BR.10 observation patients, the 15-gene signature was demonstrated to be an independent prognostic marker among all 169 DCC patients (HR 2.9, 95% Cl 1.5-5.6, p=0.002; Table 2). Subgroup analyses showed results that were not statistically significant among patients from DCC-UM (HR 1.5, 95% Cl 0.54-4.31, p=0.4; Table 2) and HLM (HR 1.2, 95% Cl 0.43-3.6, p=0.7; Table 2). The 15-gene signature was also prognostic among UM-SQ patients (HR 2.3, 95% Cl 1.1-4.7, p=0.026; Table 2), and in the Duke's patients (HR 1.5, 95% Cl 0.81-2.89, p=0.19; Table 2).

The prognostic value of the 15-gene signature was tested in stage I patients of the DCC (n=141) patients and was able to identify patients with significantly different survival outcome (Table 8). The same results apply to the 13-gene signature, given the lack of contribution of MLANA and MYT1L to the risk score (FIG. 7).

Prediction of Chemotherapy Benefit

When tested on the microarray data of 71 JBR.10 patients who received adjuvant chemotherapy, the 15-gene signature was not prognostic (HR 1.5, 95% Cl 0.7-3.3, p=0.28, Table 2). The signature was also not prognostic when applied separately to stage 1B and stage 11 patients (Table 2). Among the DCC patients, 41 were identified as having received adjuvant chemotherapy with or without radiotherapy. The 15-gene signature was also not prognostic for these 41 patients (HR 1.1, 95% Cl 0.5-2.5, p=0.8) (Table 2).

Stratified analysis showed that in JBR.10 patients with microarray data, only patients classified to the high-risk group derived benefit from the adjuvant chemotherapy (FIGS. 3C and 3D). High-risk patients showed 67% improved survival when treated by adjuvant chemotherapy compared to observation (HR=0.33, 95% Cl 0.17-0.63, p=0.0005, FIG. 3D), while those assigned to the low risk group did not benefit (FIG. 3C). These results were reproduced when applied separately to both the stage IB (FIGS. 3E and 3F) and stage II (FIGS. 3G and 3H) patients.

Multivariate analysis showed that the decrease of survival associated with adjuvant chemotherapy was independent of the stage (HR=2.26, 95% Cl 1.03-4.96, p=0.04). A Cox regression model with chemotherapy received and risk group indicator and their interaction term as independent covariates were performed to fit the overall survival data on the 133 patients with microarray data. This analysis revealed that the interaction term is highly significant (p=0.0003) with the high-risk group deriving significantly greater benefit from adjuvant chemotherapy.

The results and conclusions apply equally to the 13-gene signature.

The Initial Study Population

The initial study population comprised a subset of the patients randomized in the JBR.10 trial. There were 169 frozen tumor samples collected from patients who had their surgery at one of the BR.10 Canadian Centres and have consented to the use of their samples for “future” studies in addition to RAS mutation analysis. The samples were harvested using a standardized protocol that was agreed upon during trial protocol development by designated pathologists from each participating centre. All tumors and corresponding normal lung tissue were collected as soon as or within 30 min after resection, and were snap-frozen in liquid nitrogen. For each frozen tissue fragment, a 1 mm cross-section slice was fixed in 10% buffered formalin and submitted for paraffin embedding. Histological evaluation of the HE stained sections revealed 166 samples that contained 20% tumor cellularity. Among the latter, gene expression profiling was completed successfully in samples from 133 patients. These included 58 patients randomized to the observation (OBS) arm and 75 to the adjuvant chemotherapy (ACT) arm. However, 4 ACT patients refused chemotherapy, and for the purpose of this analysis, they were assigned to the OBS arm. Therefore, the final distribution included 62 OBS patients and 71 ACT patients (FIGS. 1 and 4).

Microarray Data Analysis

The raw microarray data from Affymetrix U133A (Affymetrix, Santa Clara, Calif.) were pre-processed using RMAexpress v0.32, then were twice log 2 transformed since the distribution of additional log 2 transformed data appeared more normal. Probe sets were annotated using NetAffx v4.2 annotation tool and only grade A level probe sets 3 (NA24) were included for further analysis. Affymetrix U133A chip contains 22,215 probe sets (19,619 probe sets with grade A annotation). Since the microarray hybridizations were performed in two batches at two separate occasions (January 2004, and June 2005), and unsupervised clustering showed that a batch difference was significant (FIG. 6), a distance-weighted discrimination (DWD) algorithm (https://genome.unc.edu/pubsup/dwd/index.html) was applied to homogenize the two batches. The DWD algorithm first finds a hyperplane that separates the two batches and adjusts the data by projecting the different batches on the DWD plane, finds the batch mean, and then subtracts out the DWD plane multiplied by this mean. In addition, the data were Z score transformed which made the validation across different datasets possible.

Univariate Analysis

The association of the expression of the individual probe set with overall survival (date of randomization to date of last follow up or death) was evaluated by Cox proportional hazards regression. The expression data for 62 patients in observation arm revealed 1312 probe sets that were associated with overall survival at p<0.05. Using a more stringent selection criteria of p<0.005, 172 probe sets with grade A annotation were prognostic.

Gene Set Signature Selection

To generate the gene expression signature, an exclusion selection procedure was firstly applied and followed by an inclusion process. The MAximizing R Square Algorithm (MARSA) included 3 sequential steps: a) probe set pre-selection; b) signature optimization; and c) leave-one-out-cross-validation. First, the candidate probe sets were pre-selected by their associations with survival at p<0.005 level. To remove the cross platform variation, expression data was z score transformed and risk score (z score weighted by the coefficient of the univariate Cox regression) was used to synthesize the information of the probe set combination. The candidate probe sets were then subjected to an exclusion followed by an inclusion selection procedure. For the preselected 172 probe sets, the exclusion procedure excluded one probe at a time, summed up the risk score of the remaining 171 probes, the calculated the R square (R², Goodness-of-fit) of the Cox model^(5, 6). Risk score was dichotomized by an outcome-orientated optimization of cutoff macro based on log-rank statistics (http://ndc.mayo.edu/mayo/research/biostat/sasmacros.cfm) before being introduced to the Cox proportional hazards model. A probe set was excluded if its exclusion resulted in obtaining the largest R². The procedure was repeated until there was only one probe set left. An inclusion procedure was followed using the probe set left by the exclusion procedure as the starting probe set. It included one probe set at a time, summed up the risk score of the included probe sets and risk score was dichotomized and R² was calculated. The probe set was included if its inclusion resulted in obtaining the largest R². The exclusion procedure produced a largest R square of 0.67 by a minimal 7 probe combination and the inclusion procedure generated a largest R² of 0.78 by a 15 probe combination (FIG. 1B). Then, the 15-gene signature disclosed in WO 2009/137921, published on Nov. 19, 2009, was established after passing the internal validation by leave-one-out-cross-validation (LOOCV) and external validation on other datasets (listed below). All statistical analyses were performed using SAS v9.1 (SAS Institute, Calif.). The 13-gene signature of the present disclosure proved to perform as well as the 15-gene signature (FIG. 7; Table 4).

Prognostic Modeling by Principal Component Analysis of Signature Genes

Principal components analysis (PCA) (based on correlation matrix) was carried out to synthesize the information across the chosen gene probe sets and reduce the number of covariates in building the prognostic model. The eigenvalue of greater than or equal to 1 was used as cutoff point in determining how many proponents to include in the model, and those significantly correlated to disease-specific survival (DSS) were included in the final multivariable model. The PCA analysis was done based on all 133 patients with microarray data. When correlated to the DSS based on the 62 observation patients, the first 4 principal components were found to satisfy the criteria and were included in the prognostic model. Table 10 lists the four principal components for each of the 13 genes in the 13-gene signature. The same analysis can be applied to derive principal component coefficients for additional genes selected from the 172 genes listed in Table 3, such as for example, RGS4, UGT2B4, and/or MCF2. Furthermore, one of skill will appreciate from the above description how to obtain the first four principal component coefficients for any of the genes listed in Table 3.

To determine the gene signature prognostic group, multivariate Cox regression model with the first 4 principal components were fitted to the disease specific survival of the 62 observation patients. The linear prognostic scores were calculated by the sum of the multiplication of the estimated coefficient from Cox model and the corresponding principal component value. Using the prognostic score, patients were divided into low and high risk group based on the median of the prognostic score, i.e., those with prognostic score less than the median as low risk group, while those with score no less than the median as high risk group. For the 62 observation patients with microarray data, 31 patients were classified in each group. Applying the same rule to the 71 chemo-treated patients, 35 patients were classified in low risk group and 36 patients in high-risk group.

Validation of General Applicability of Prognostic Signature

Validation of the 15-gene signature was carried out on stage I-II cases from Duke, UM-SQ, and DCC who did not receive adjuvant chemotherapy. When the risk score was dichotomized using the cutoff determined from the BR.10 training set, the 15-gene signature was able to separate 38 cases of low risk from 47 cases of high risk (log rank p=0.226) of NSCLC in the Duke dataset. Multivariate analysis (adjusted for stage, histology and patients' age and gender) showed that the 15-gene signature was an independent prognostic factor (HR=1.5, 95% Cl 0.81-2.89, p=0.19, Table 2). Raponi contains squamous cell carcinoma only and the cases have the worst survival rate. However, the 15-gene signature was still able to separate 50 cases of low risk from 56 cases with high risk (log rank p=0.0447) and this separation was independent of stage and patients' age and gender (HR=2.3, 95% Cl 1.1-4.7 p=0.026, Table 2). The DCC dataset contained only adenocarcinoma cases. Applying the 15-gene signature on DCC stage I and II, was able to separate 87 low risk cases from the 82 high risk cases (log rank p=0.0002, FIG. 2E). Multivariate analysis (adjusted for stage and patients' age and gender) showed that the prognostic value of the 15-gene signature was independent prognostic factor (HR=2.9, 95% Cl 1.5-5.6, p=0.002, Table 2). There were 67 stage IB-II cases without chemotherapy in MI, the 15-gene signature was able to separate 44 low risk cases from the 23 high risk cases (log rank p=0.013). Multivariate analysis (adjusted for stage and patients' age and gender) showed that the prognostic value of the 15-gene signature was independent prognostic factor (HR=1.5, 95% Cl 0.54-4.31, p=0.4, Table 2). Cases from MSKCC had a significantly better 5-year overall survival compared to other datasets. However, the 15-gene signature was able to separate 32 cases of low risk from 32 cases of high risk in MSKCC (log rank p=0.16). Multivariate analysis (adjusted for stage) revealed that the 15-gene signature was an independent prognostic factor. Validation of the 15-gene signature on HLM revealed that the 15-gene signature was able to separate 26 cases of low risk from 24 cases of high risk (log rank p=0.0084). Multivariate analysis (adjusted for stage) showed that there was a trend to separation by the 15-gene signature (HR=1.2, 95% Cl 0.43-3.6, p=0.7). These validation data confirm that the 15-gene signature is a strong prognostic signature and its power of predicting the outcome of NSCLC is independent of and superior to that of stage. Since MLANA and MYT1L proved to have no significant effect on the risk score (FIG. 7), validation data thus confirm that the 13-gene signature is a strong prognostic signature and its power of predicting the outcome of NSCLC is independent of and superior to that of stage.

The Benefit of Chemotherapy was Limited to High Risk Patients

A total of 30 deaths were observed in the ACT. Six of them were due to other malignancies. The 15-gene signature was unable to separate the good/bad outcome patients (p=0.83, data not shown) in the ACT. However, stratified analysis showed that only patients with high risk derived benefit from adjuvant chemotherapy (FIG. 3D). Upon receiving adjuvant chemotherapy, the survival rate of the 36 high-risk patients was significantly improved (HR=0.33, 95% Cl 0.17-0.63, p=0.0005, FIG. 3D). On the other hand, the application of chemotherapy on low risk patients resulted in a decrease in survival rate (HR=3.67, 95% Cl 1.22-11.06, p=0.0133, FIG. 3C). Death was evenly distributed between the low and high risk groups in the ACT arm (15 deaths in low and high risk group, respectively). Each of these two groups contained 3 deaths that were not due to lung cancer. Stratification by risk group and stage showed that the survival rate of high risk patients from both stage IB and stage II was significantly improved by chemotherapy (FIGS. 3F and H). Moreover, for low risk patients of stage II, chemotherapy was associated with significantly decreased survival (FIGS. 3E and G). A Cox regression model with chemotherapy received and risk group indicator and their interaction term as independent covariates was performed to fit the overall survival data on the 133 patients with microarray data. This analysis revealed that the interaction term is highly significant (p=0.0002) with the high-risk group deriving significantly greater benefit from adjuvant chemotherapy.

Discussion:

Gene expression signature is thought to represent the altered key pathways in carcinogenesis and thus is able to predict patients' outcome. However, being able to faithfully represent the altered key pathways, the signature must be generated from genome-wide gene expression data. The present study used all information generated by Affymetrix U133A chip on NSCLC samples from a randomized clinical trial to derive a 15-gene signature. The 15-gene signature was able to identify 50% (31/62) stage IB-II NSCLC patients had relative good outcome. Multivariate analysis indicated that the 15-gene signature was an independent prognostic factor. Moreover, its independent prognostic effect had been in silico validated on 169 adenocarcinomas without adjuvant chemo- or radio-therapy from DCC and 85 NSCLC from Duke and 106 squamous cell carcinomas of the lung from the University of Michigan (UM-SQ). Importantly, the 15-gene signature was able to predict the response to adjuvant chemotherapy with high-risk patients across the stages being benefited from adjuvant chemotherapy. This finding was also validated on DCC dataset.

When attempting to migrate the detection platform to quantitative PCR, two genes (MLANA and MYT1L) were difficult to detect reproducably, and after further analysis were discovered to have no significant effect on the risk score (FIG. 7). Thus, a 13-gene signature also predicts the response to adjuvant chemotherapy with high-risk patients across the stages being benefited from adjuvant chemotherapy.

Adjuvant chemotherapy for completely resected early stage NSCLC was a research question until the results of a series of positive trials^(2, 4), including BR.10³, were published. However, whether chemotherapy played a beneficial role in stage IB remained to be clarified²⁻⁶. The present study showed that the stage 1B patients were potentially able to be separated into low (49.3%, 36/73) and high (50.7%, 37/73) risk groups using the 15-gene signature. Upon administering the adjuvant chemotherapy to stage IB patients, the survival rate of patients with high risk was significantly improved (p=0.0698, FIG. 3F) whereas patients with low risk did not experience a benefit in survival (p=0.0758, FIG. 3E). Therefore the effect of chemotherapy on stage IB NSCLC was neutralized and thus gave an incorrect impression that no beneficial effect existed³. Based on the evidence provided here and from the meta-analysis⁶, it may be concluded that 50.7% (37/73) stage IB NSCLC patients have the potential to benefit from adjuvant chemotherapy. Since MLANA and MYTL1 proved to have no significant effect on the risk score (FIG. 7), the 13-gene signature is also a predictor that 50.7% (37/73) stage IB NSCLC patients have the potential to benefit from adjuvant chemotherapy.

Another significance of the present study was that the signature was able to identify a subgroup (50%, 30/60) of patients from stage 11 who did not benefit from adjuvant chemotherapy (p=0.1498, FIG. 3G). In current practice, adjuvant chemotherapy is recommended for all patients. However, the 15-gene signature suggests that about a half of the stage II patients may not benefit from adjuvant chemotherapy. Since MLANA and MYT1L have no significant effect on the risk score (FIG. 7), the 13-gene signature also serves as a basis to conclude that about half of the stage II patients may not benefit from adjuvant chemotherapy.

The gene ontology analysis showed that in the 13-gene signature, 4 genes (FOSL2, HEXIM1, IKBKAP, and ZNF236) were involved in the regulation of transcription. EDN3 and STMN2 played a role in signal transduction. Transformed 3T3 cell double minute 2 (MDM2), an E3 ubiquitin ligase, which targets p53 protein for degradation, plays a key role in cell cycle and apoptosis. Dworakowska D. et al.²⁴ reported that overexpression of MDM2 protein was correlated with low apoptotic index, which was associated with poorer survival. Myoglobin (MB) palyed a role in response to hypoxia and Uridine monophosphate synthetase (UMPS) participated in the ‘de novo’ pyrimidine base biosynthetic process, however, none of them has not been explored in lung cancer. The L1 cell adhesion molecule (L1CAM) involved in cell adhesion whose overexpression was associated with tumor metastasis and poor prognosis²⁵⁻²⁸. ATPase, Na+/K+ transporting, beta 1 polypeptide (ATP1B1) was involved in ion transport which was reported recently to be able to discriminate the serous low malignant potential and invasive epithelial ovarian tumors²⁹. These findings indicated that cellular transcription, cell cycle and apoptosis, cell adhesion and response to hypoxia were important for lung cancer progression.

The range of expression levels of members of the 13-gene signature was broad, from very low expression level such as MDM2 and ZNF236 to fairly high expression such as TRIM14 or very high expression such as ATP1B1 (Table 4). Least variable gene (<5%), such as UMPS (Table 4), was also a member of the signature. These data suggested that it may not be a good practice to exclude low expressed and least variable probe set in the data pre-selection process in an arbitrary way. The signature generated using the present strategy performed better than that of Raponi's method of using the top 50 genes. There are only 3 genes (IKBKAP, L1CAM, and FAM64A) whose significance in association with survival is in the top 50 genes (Table 4).

Materials and Methods: Patients and Samples

Included in the JBR.10 protocol was the collection of snap-frozen or formalin-fixed paraffin embedded tumor samples for KRAS mutation analysis and tissue banking for future laboratory studies³. Altogether 445 of 482 randomized patients consented to banking. Snap-frozen tissues were collected from 169 Canadian patients (FIG. 4). Histological evaluation of the HE section from the snap-frozen tumor samples revealed 166 that contained an estimated >20% tumor cellularity; gene expression profiling was completed in 133 of these patient samples, using the U133A oligonucleotide microarrays (Affymetrix, Santa Clara, Calif.). Profiling was not completed in 33 patient samples. Of 133 patients with microarray profiles, 62 did not received post-operative adjuvant chemotherapy and were group as observation patients, while 71 patients were received chemotherapy. University Health Network Research Ethics Board approved the study protocol.

RNA Isolation and Microarray Profiling

Total RNA was isolated from frozen tumor samples after homogenization in guanidium isothiocyanate solution and acid phenol-chloroform extraction. The quality of isolated RNA was assessed initially by gel electrophoresis, followed by the Agilent Bioanalyzer. Ten micrograms of total RNA was processed, labeled, and hybridized to Affymetrix's HG-U133A GeneChips. Microarray hybridization was performed at the Center for Cancer Genome Discovery of Dana Farber Cancer Institute.

Microarray Data Analysis and Gene Annotation

The raw microarray data were pre-processed using RMAexpress v0.3²². Probe sets were annotated using NetAffx v4.2 annotation tool and only grade A level probe sets²³ (NA22) were included for further analysis. Because the microarray profiling was done in two separate batches at different times and unsupervised heuristic K-means clustering identified a systematic difference between the two batches (FIG. 6), the distance-weighted discrimination (DWD) method (https://genome.unc.edu/pubsup/dwd/index html) was used to adjust the difference. The DWD method first finds a separating hyperplane between the two batches and adjusts the data by projecting the different batches on the DWD plane, discover the batch mean, and then subtracts out the DWD plane multiplied by this mean. The data were then transformed to Z score by centering to its mean and scaling to its standard deviation. This transformation was necessary for validation on different datasets in which different expression ranges are likely to exist, and for validation on different platforms, such as qPCR where the data scale is different.

Derivation of Signature

The pre-selected probe sets by univariate analysis at p<0.005 were selected by an exclusion procedure. The exclusion selection excluded one probe set at a time based on the resultant R square (R², Goodness-of-fit^(15, 16)) of the Cox model. It kept repeating until there was only one probe set left. The procedure was repeated until there was only one probe set left. An inclusion procedure was followed using the probe set left by the exclusion procedure as the starting probe set. It included one probe set at a time based on the resultant R² of the Cox model. Finally, the R² was plotted against the probe set and a set of minimum number of probe sets yet having the largest R² was chosen as candidate signature. Gene signature was established after passing the internal validation by leave-one-out-cross-validation (LOOCV) and external validation on other datasets (listed below). All statistical analyses were performed using SAS v9.1 (SAS Institute, Calif.).

Validation in Separate Microarray Datasets

The prognostic value of this 15-gene signature was tested on separate microarray datasets. Three represented subsets of microarray data from the NCl Director's Challenge Consortium (DCC) for the Molecular Classification of Lung Adenocarcinoma (Nature Medicine, in review/in press). In total, the Consortium analyzed the profiles of 442 tumors, including 177 from University of Michigan (UM), 79 from H. L. Moffitt Cancer Centre (HLM), 104 from Memorial Sloan-Kettering Cancer Centre (MSK), and 82 from our group. As 39 of the latter tumors overlap with samples used in this study, only data from the first 3 groups were used for validation. In addition, patients who were noted as either unknown or having received adjuvant chemotherapy and/or radiotherapy were excluded. Therefore, the DCC dataset used in this validation study included only 169 patients: 67 from UM, 46 from HLM, 56 from MSK. Two additional published microarray datasets were also used for validation: the Duke's University dataset of 85 non-small cell lung cancer patients (Potti, et al, NEJM), and the University of Michigan dataset of 106 squamous cell carcinomas patients (UM-SQ) (Raponi et al). Raw data of these microarray studies were downloaded and RMA pre-processed. The expression levels were Z score transformed after double log 2 transformation. Risk score was the Z score weighted by the coefficient of the Cox model from the OBS. Demographic data of the DCC cohort was listed in Table 5.

The prognostic value of 2 genes, MLANA and MYT1L was tested on the same microarray datasets. The DCC dataset used in this validation study included 169 patients: 67 from UM, 46 from HLM, 56 from MSK. Two additional published microarray datasets were also used for validation: the Duke's University dataset of 85 non-small cell lung cancer patients (Potti, et al, NEJM), and the University of Michigan dataset of 106 squamous cell carcinomas patients (UM-SQ) (Raponi et al). Raw data of these microarray studies were downloaded and RMA pre-processed. The expression levels were Z score transformed after double log 2 transformation. FIG. 7 represents the lack of these 2 genes to contribute significantly to the signature.

Statistical Analysis

Risk score was the product of coefficient of Cox proportional model and the standardized expression level. The univariate association of the expression of the individual probe set with overall survival (date of randomization to date of last followup or death) was evaluated by Cox proportional hazards regression. A stringent p<0.005 was set as a selection criteria in order to minimize the possibility of false-positive results.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Example 2

This application describes a gene expression-based prognostic signature in early-stage (stage I and II) non-small-cell lung cancer (NSCLC) for identifying, more selectively than the current clinicopathological criteria (i.e. TNM staging, gender, age, etc.), patients with significantly different prognoses. The expression of signature genes is measured from a patient's tumour sample and converted into a risk score which then classifies the patient into distinct prognostic risk groups. The higher/lower risk of mortality stratification of patients is independent of clinicopathological criteria, including stage, histology, gender and age, and may assist in guiding the post-surgical treatment of early-stage NSCLC patients, i.e. suggesting chemotherapy for higher risk stage I patients but avoiding it for stage II patients with a lower risk profile.

Reverse transcription quantitative PCR (RT-qPCR) may have advantages for clinical settings, and moreover, the majority of NSCLC tumour specimens resected in hospitals are fixed in formalin and subsequently embedded in paraffin (formalin-fixed, paraffin-embedded: FFPE). This preservation method also allows for long-term storage of the tissue samples. Therefore, if the signatures could be migrated to an RT-qPCR platform and used with RNA derived from FFPE-preserved NSCLC specimens, the test may be more easily integrated into the clinical process.

This example describes the validation of a gene signature to fresh frozen and FFPE samples, using a quantitative RT-PCR platform as compared to a microarray platform.

Materials and Methods Tissue Samples

Resected tumour samples from untreated stage I and stage II NSCLC patients, fixed in formalin and embedded in paraffin (FFPE) were secured from the Ontario Tumour Bank (OTB) in Toronto and the Ohio State University as part of the Cooperative Human Tissue Network (CHTN). In addition, a set of matched fresh frozen and FFPE-preserved NSCLC tissue specimens was received from OTB.

Total RNA Preparation

Total RNA was isolated from FFPE-tissue sections as curls or mounted onto slides using the RecoverAll kit following standard procedures (Ambion). After elution in water, the total RNA concentration was assessed with the 260/280 nm ratio using the NanoDrop spectrophotometer (Thermo Scientific). If the concentration was below 70 ng/μL the total RNA was concentrated using Amicon Ultra Centrifugal Filters (Millipore). The quality of the isolated total RNA was evaluated using the 2100 Bioanalyzer (Agilent Technologies). RNA integrity numbers (RIN) between 2-2.5 were detected. RNA from a cohort of matched frozen tissue specimens was isolated using the RNeasy Plus Mini kit (Qiagen) and subjected to the same methods to assess quantity and quality.

ABI TaciMan® Assays

TaqMan® gene expression assays (Applied Biosystems) suitable for the detection of RNA derived from FFPE-preserved tissue samples were selected for the 15 signature genes. Five previously published reference genes for the use in either lung cancer and/or gene expression from FFPE tissue samples were selected (BAT1 and TBP (Barsyte-Lovejoy, Lau et al. 2006), ESD (Saviozzi, Cordero et al. 2006), IPO8 (Nguewa, Agorreta et al. 2008), and TFRC (Drury, Anderson et al. 2009)).

Custom Designed Gene Expression Assays

Up to six hydrolysis probe-based assays optimized for the detection of RNA from FFPE-preserved and fresh frozen tumor samples were designed for each of the 15 signature genes (IDT Technologies and Roche Applied Science).

cDNA Synthesis, cDNA Amplification and PCR Analysis

Total RNA was reverse transcribed using the iScript cDNA Synthesis kit according to the manufacturer's protocol (BioRad). The TaqMan® PreAmp Master Mix kit (Applied Biosystems) was used to amplify 12.5 ng of respective cDNA with gene-specific assays according the manufacturer's protocol. The real-time PCR reactions were on the 7900 HT Fast Real-Time PCR System (Applied Biosystems, 384 well plate format) in a total reaction volume of 12 μl.

Normalization

The reactions were run in quadruplicates and the relative expression was calibrated on either a pancreas (only for the gene EDN3) or a universal human reference RNA (UHRR) standard curve. To normalize the expression of the genes in the signature, the geometric mean of the selected reference genes for either frozen or FFPE-derived RNA was used.

Identification of Reference Genes

The GeNorm (Vandesompele, De Preter et al. 2002) and NormFinder (Andersen, Jensen et al. 2004) algorithms were used to determine the most suitable (most stable and least variable) combination of reference genes for the use with either fresh frozen or FFPE-derived RNA.

Statistical Analysis

The intraclass correlation (ICC) (Shrout and Fleiss 1979) was used to assess the agreement of the two risk score calculations. Values of this statistic range from −1 to 1, with 1 indicating exact agreement. Intermediate values of agreement were defined by Landis and Koch (1977) as following: Moderate (0.41-0.60), substantial (0.61-0.80), and almost perfect (0.81-1.00) (Landis and Koch 1977).

Results

Testing Pre-Validated ABI Assays on RNA Derived from FFPE-Preserved NSCLC Tissue Samples

Pre-validated TaqMan assays for each of the 15 signature genes were run on cDNA synthesized from the total RNA of 20 FFPE-preserved NSCLC tissue specimens. The most stable reference gene, TBP, was identified using the NormFinder algorithm from a set of previously published references genes for the use with either NSCLC or FFPE. Normalization using TBP and calculation of the relative gene expression revealed that 13 of the 15 signature genes were reliably detectable across a set of 20 different FFPE NSCLC tissue samples.

Up to 6 hydrolysis-based assays were designed for each of the 15 signature genes for the use with RNA derived from FFPE-preserved samples (amplicon size <100 nt) using two different probe chemistries (i.e., regular TaqMan assays or locked nucleic acid assays (LNA). These assays were run on serially diluted cDNA derived from FFPE-preserved or fresh frozen human reference RNA (UHRR, or pancreas RNA (EDN3)). Standard curves were generated to estimate the PCR efficiency and R2 to select the best assay for each gene. An assay was chosen if the Ct value was below 36, the PCR efficiency was between 90-110% and R2>0.8 (Table 15). For EDN3 only one assay of the six designed assays showed sufficient performance on FFPE samples, with a slightly higher PCR efficiency (116%).

An analysis of in silico microarray data revealed that the two genes MYT1L and MLANA did not significantly contribute to the risk score to predict outcome. When the C-index with and without these two genes was calculated and compared (FIG. 7), there was no statistically significant difference between the risk score calculated from the 13 or the 15 genes. All subsequent experiments were therefore performed with 13 signature genes.

Selection of Normalization Genes and Testing Custom Assays on a Set of Matched FFPE and Frozen NSCLC Samples

A set of matched frozen and FFPE-preserved early-stage NSCLC samples were secured from the Ontario Tumour Bank to select reference gene(s) for use with RNA derived from FFPE or fresh frozen preserved NSCLC tissue specimens and to test the reliability of the custom assays in detecting gene expression. To identify the best reference genes for use with RNA derived from FFPE or fresh frozen lung cancer samples, the expression of 14 previously published reference genes was evaluated with the GeNorm algorithm. A group of four reference genes (“normalization genes”) was found to be stable and substantially lacking in variable for the use with RNA derived from FFPE (BAT1, TBP, PP1A and GUSB) or frozen NSCLC samples (BAT1, TBP, PP1A and 1P08). Three of the four genes overlapped (BAT1, TBP and PP1A) (FIG. 8).

After successful identification of normalization genes and normalization to adjust for input differences, the relative gene expression of the signature genes was calculated for both the frozen and FFPE sample sets. Pearson correlation analysis was then performed to measure the linear dependence between the FFPE and frozen expression values of the matched samples. A Pearson correlation coefficient of 0.75 was found in 13 of the 18 matched NSCLC samples (FIG. 9).

Risk Score Calculations Based on RT-qPCR Expression Data and Correlation with Microarray Classifier

A strong correlation was found on a gene by gene basis between fresh frozen and FFPE-preserved tissue samples. To examine the predictive power, the risk score was calculated, using expression data from total RNA from 30 frozen tissue samples from the UHN183 cohort. RT-qPCR analysis on this sample subset was performed using the custom assays and the reference genes selected for the use with frozen tissue specimens. After normalization, the risk scores were calculated and correlated to the risk scores calculated from the previous microarray analysis. An interclass correlation coefficient (ICC) of 0.66 was found, indicating substantial correlation between risk scores calculated based on microarray and RT-qPCR data. Association with clinical outcome data and univariate survival analysis revealed a hazard ratio (HR, high risk versus low risk) of 1.76 for those 30 samples analyzed on RT-qPCR and a HR of 2.1 for the same sample set analyzed with microarray. The p values were not significant in either case due to the small sample size. The algorithm was the same as used in Example 1.

Discussion

As shown herein, gene expression detected with custom designed assays carry prognostic information for both FFPE- and frozen-preserved tissues. Furthermore, risk scores calculated from RT-qPCR-derived expression data correlate substantially with the risk scores calculated from microarray data and are useful to provide a risk classification score for mortality stratification in NSCLC patients.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

TABLE 1 Baseline factors of BR.10 patients with and without microarray profiles No Microarray microarray All profiled profiled Patients (n = 133) (n = 349) Factor (n = 482) n % n % P value Treatment received ACT 231 71 53% 160 46% 0.14 OBS 251 62 47% 189 54% Age  <65 324 87 65% 237 68% 0.6 ≧65 158 46 35% 112 32% Gender Male 314 91 68% 223 64% 0.35 Female 168 42 32% 126 36% Performance Status 0 236 67 50% 169 49% 0.72 1 245 66 50% 179 51% Stage of Disease IB 219 73 55% 146 42% 0.01 II 263 60 45% 203 58% Surgery Pneumonectomy 113 33 25% 80 23% 0.66 Other Resection 369 100 75% 269 77% Pathologic type Adenocarcinoma 256 71 53% 185 53% 0.56 Squamous 179 52 39% 127 36% Other 47 10 8% 37 11% Ras Mutation Status Present 117 28 21% 89 26% 0.12* Absent 333 105 79% 228 65% Unknown 32 0 0% 32 9% *P-value: Without include those missing or unknown.

TABLE 2 Comparison of 5-yr Survival (multivariate) of High and Low Risk Groups in Untreated Patients and Patients who Received Adjuvant Chemotherapy. n HR* 95% CI p value Observation/untreated Patients JBR.10 (randomized with 62 18.0  5.8-56.1 <0.0001 microarray) Stage IB 34 29.9  4.5-197.4 0.0004 Stage II 28 16.4  3.0-88.1 0.001 DCC (no adjuvant 169 2.9 1.5-5.6 0.002 therapy) UM 67 1.5 0.54-4.31 0.4 HLM 46 1.2 0.43-3.60 0.7 MSK 56 NA** NA Duke 85 1.5 0.81-2.89 0.19 UM-Squamous 106 2.3 1.1-4.7 0.026 Patients Treated With Adjuvant Chemotherapy BR.10 (randomized with 71 1.5 0.7-3.3 0.28 microarray) BR.10 Stage I 39 1.7 0.5-5.6 0.36 BR.10 Stage II 32 1.2 0.4-3.6 0.8 DCC (not randomized) 41 1.1 0.5-2.5 0.8 n: number of patients; HR: hazard ratio; CI: confidence interval *HR compares the survival of the poor prognostic group to that of the good prognostic group as determined by the 15-gene signature with the adjustment of stage and patients' age and gender. For BR.10, and Duke, the effect of histology was also adjusted **All events were in high risk group and female patients.

TABLE 3 172 U133A probe sets that were prognostic at p < 0.005 for the 62 BR.10 observation arm patients. Representative p Probe Set ID Public ID UniGene ID Gene Symbol Coefficients HR HRL HRH value 200878_at AF052094 Hs.468410 EPAS1 −0.58 0.56 0.37 0.84 0.0048 201228_s_at NM_006321 Hs.31387 ARIH2 0.47 1.60 1.17 2.18 0.0029 201242_s_at BC000006 Hs.291196 ATP1B1 −0.69 0.50 0.35 0.71 0.0001 201243_s_at NM_001677 Hs.291196 ATP1B1 −0.54 0.58 0.41 0.83 0.0028 201301_s_at NM_001153 Hs.422986 ANXA4 −0.55 0.58 0.40 0.83 0.0028 201502_s_at NM_020529 Hs.81328 NFKBIA −0.62 0.54 0.36 0.79 0.0016 202023_at NM_004428 Hs.516664 EFNA1 −0.67 0.51 0.35 0.76 0.0009 202035_s_at AF017987 Hs.213424 SFRP1 0.69 1.99 1.39 2.86 0.0002 202036_s_at AF017987 Hs.213424 SFRP1 0.84 2.31 1.56 3.44 0.0000 202037_s_at AF017987 Hs.213424 SFRP1 0.74 2.09 1.43 3.07 0.0002 202490_at AF153419 Hs.494738 IKBKAP 0.42 1.53 1.17 1.99 0.0018 202707_at NM_000373 Hs.2057 UMPS 0.60 1.81 1.24 2.66 0.0023 202814_s_at NM_006460 Hs.15299 HEXIM1 0.59 1.80 1.20 2.70 0.0045 203001_s_at NM_007029 Hs.521651 STMN2 0.55 1.73 1.21 2.47 0.0027 203147_s_at NM_014788 Hs.575631 TRIM14 −0.56 0.57 0.39 0.82 0.0028 203438_at AI435828 Hs.233160 STC2 0.67 1.96 1.29 2.96 0.0015 203444_s_at NM_004739 Hs.173043 MTA2 0.38 1.46 1.12 1.89 0.0046 203475_at NM_000103 Hs.511367 CYP19A1 0.56 1.76 1.23 2.52 0.0021 203509_at NM_003105 Hs.368592 SORL1 −0.58 0.56 0.39 0.81 0.0020 203928_x_at AI870749 Hs.101174 MAPT 0.44 1.55 1.15 2.10 0.0044 203973_s_at M83667 Hs.440829 CEBPD −0.61 0.54 0.38 0.77 0.0005 204179_at NM_005368 Hs.517586 MB 0.47 1.60 1.16 2.22 0.0044 204267_x_at NM_004203 Hs.77783 PKMYT1 0.63 1.87 1.28 2.73 0.0011 204338_s_at AL514445 Hs.386726 RGS4 0.57 1.77 1.23 2.53 0.0021 204531_s_at NM_007295 Hs.194143 BRCA1 0.60 1.82 1.21 2.75 0.0043 204584_at AI653981 Hs.522818 L1CAM 0.56 1.75 1.30 2.35 0.0002 204684_at NM_002522 Hs.645265 NPTX1 0.48 1.61 1.18 2.19 0.0024 204810_s_at NM_001824 Hs.334347 CKM 0.46 1.58 1.20 2.09 0.0012 204817_at NM_012291 — ESPL1 0.53 1.70 1.24 2.34 0.0010 204933_s_at BF433902 Hs.81791 TNFRSF11B 0.51 1.67 1.27 2.20 0.0003 204953_at NM_014841 Hs.368046 SNAP91 0.59 1.81 1.31 2.49 0.0003 205046_at NM_001813 Hs.75573 CENPE 0.62 1.86 1.28 2.70 0.0012 205189_s_at NM_000136 Hs.494529 FANCC 0.53 1.70 1.21 2.40 0.0023 205217_at NM_004085 Hs.447877 TIMM8A 0.64 1.90 1.26 2.85 0.0020 205386_s_at NM_002392 Hs.567303 MDM2 0.49 1.63 1.19 2.23 0.0025 205433_at NM_000055 Hs.420483 BCHE 0.58 1.79 1.23 2.62 0.0024 205481_at NM_000674 Hs.77867 ADORA1 0.49 1.63 1.20 2.23 0.0020 205491_s_at NM_024009 Hs.522561 GJB3 0.46 1.58 1.18 2.11 0.0021 205501_at AI143879 Hs.348762 — 0.40 1.49 1.13 1.97 0.0043 205825_at NM_000439 Hs.78977 PCSK1 0.59 1.81 1.24 2.65 0.0023 205893_at NM_014932 Hs.478289 NLGN1 0.40 1.49 1.13 1.97 0.0048 205938_at NM_014906 Hs.245044 PPM1E 0.52 1.68 1.22 2.31 0.0013 205946_at NM_003382 Hs.490817 VIPR2 0.50 1.65 1.17 2.33 0.0043 206043_s_at NM_014861 Hs.6168 ATP2C2 −0.55 0.57 0.39 0.84 0.0044 206096_at AI809774 Hs.288658 ZNF35 0.55 1.73 1.20 2.49 0.0034 206228_at AW769732 Hs.155644 PAX2 0.50 1.65 1.27 2.15 0.0002 206232_s_at NM_004775 Hs.591063 B4GALT6 0.44 1.56 1.17 2.07 0.0021 206401_s_at J03778 Hs.101174 MAPT 0.39 1.48 1.13 1.94 0.0049 206426_at NM_005511 Hs.154069 MLANA 0.63 1.87 1.26 2.77 0.0018 206496_at NM_006894 Hs.445350 FMO3 0.53 1.70 1.22 2.37 0.0018 206505_at NM_021139 Hs.285887 UGT2B4 0.61 1.84 1.26 2.69 0.0017 206524_at NM_003181 Hs.389457 T 0.78 2.18 1.35 3.53 0.0015 206552_s_at NM_003182 Hs.2563 TAC1 0.97 2.63 1.53 4.53 0.0005 206619_at NM_014420 Hs.159311 DKK4 0.54 1.72 1.20 2.45 0.0029 206622_at NM_007117 Hs.182231 TRH 0.53 1.70 1.23 2.37 0.0015 206661_at NM_025104 Hs.369998 DBF4B 0.55 1.73 1.27 2.36 0.0005 206672_at NM_000486 Hs.130730 AQP2 0.37 1.45 1.13 1.84 0.0030 206678_at NM_000806 Hs.175934 GABRA1 0.39 1.48 1.16 1.89 0.0014 206799_at NM_006551 Hs.204096 SCGB1D2 0.41 1.51 1.15 1.99 0.0032 206835_at NM_003154 Hs.250959 STATH 0.46 1.59 1.16 2.18 0.0042 206940_s_at NM_006237 Hs.493062 POU4F1 0.54 1.72 1.23 2.40 0.0017 206984_s_at NM_002930 Hs.464985 RIT2 0.47 1.59 1.16 2.20 0.0045 207003_at NM_002098 Hs.778 GUCA2A 0.62 1.85 1.23 2.79 0.0032 207028_at NM_006316 Hs.651453 MYCNOS 0.48 1.61 1.19 2.18 0.0020 207208_at NM_014469 Hs.121605 HNRNPG-T 0.51 1.66 1.23 2.26 0.0010 207219_at NM_023070 Hs.133034 ZNF643 0.60 1.82 1.27 2.60 0.0011 207529_at NM_021010 — DEFA5 0.65 1.91 1.38 2.64 0.0001 207597_at NM_014237 Hs.127930 ADAM18 0.63 1.87 1.36 2.58 0.0001 207814_at NM_001926 Hs.711 DEFA6 0.61 1.85 1.21 2.81 0.0041 207843_x_at NM_001914 Hs.465413 CYB5A −0.55 0.58 0.39 0.84 0.0047 207878_at NM_015848 — KRT76 0.41 1.51 1.17 1.95 0.0017 207937_x_at NM_023110 Hs.264887 FGFR1 0.43 1.54 1.14 2.08 0.0045 208157_at NM_009586 Hs.146186 SIM2 0.45 1.56 1.19 2.05 0.0013 208233_at NM_013317 Hs.468675 PDPN 0.54 1.72 1.18 2.49 0.0043 208292_at NM_014482 Hs.158317 BMP10 0.44 1.55 1.17 2.05 0.0025 208314_at NM_006583 Hs.352262 RRH 0.56 1.75 1.19 2.58 0.0044 208368_s_at NM_000059 Hs.34012 BRCA2 0.62 1.86 1.26 2.73 0.0018 208399_s_at NM_000114 Hs.1408 EDN3 0.48 1.61 1.18 2.20 0.0028 208511_at NM_021000 Hs.647156 PTTG3 0.49 1.63 1.17 2.29 0.0043 208684_at U24105 Hs.162121 COPA −0.52 0.59 0.41 0.85 0.0041 208992_s_at BC000627 Hs.463059 STAT3 −0.67 0.51 0.34 0.77 0.0012 209434_s_at U00238 — PPAT 0.43 1.54 1.15 2.06 0.0033 209839_at AL136712 Hs.584880 DNM3 0.54 1.72 1.18 2.50 0.0049 209859_at AF220036 Hs.368928 TRIM9 0.45 1.57 1.16 2.12 0.0032 210016_at BF223003 Hs.434418 MYT1L 0.60 1.82 1.31 2.52 0.0003 210247_at AW139618 Hs.445503 SYN2 0.64 1.89 1.30 2.75 0.0008 210302_s_at AF262032 Hs.584852 MAB21L2 0.59 1.81 1.34 2.44 0.0001 210315_at AF077737 Hs.445503 SYN2 0.66 1.94 1.31 2.87 0.0009 210455_at AF050198 Hs.419800 C10orf28 0.57 1.76 1.24 2.50 0.0015 210758_at AF098482 Hs.493516 PSIP1 0.42 1.52 1.17 1.97 0.0015 210918_at AF130075 — — 0.46 1.59 1.24 2.04 0.0003 211204_at L34035 Hs.21160 ME1 0.54 1.72 1.26 2.33 0.0006 211264_at M81882 Hs.231829 GAD2 0.53 1.71 1.19 2.44 0.0034 211341_at L20433 Hs.493062 POU4F1 0.57 1.77 1.21 2.58 0.0031 211516_at M96651 Hs.68876 IL5RA 0.60 1.82 1.26 2.62 0.0013 211772_x_at BC006114 Hs.89605 CHRNA3 0.52 1.69 1.22 2.33 0.0014 212359_s_at W89120 Hs.65135 KIAA0913 −0.53 0.59 0.42 0.82 0.0019 212528_at AI348009 Hs.633087 — −0.79 0.45 0.29 0.70 0.0004 212531_at NM_005564 Hs.204238 LCN2 −0.57 0.56 0.38 0.84 0.0049 213197_at AB006627 Hs.495897 ASTN1 0.66 1.93 1.36 2.74 0.0002 213260_at AU145890 Hs.599993 — 0.51 1.67 1.18 2.35 0.0036 213458_at AB023191 — KIAA0974 0.43 1.54 1.19 1.99 0.0010 213482_at BF593175 Hs.476284 DOCK3 0.53 1.70 1.19 2.42 0.0032 213603_s_at BE138888 Hs.517601 RAC2 −0.62 0.54 0.37 0.79 0.0017 213917_at BE465829 Hs.469728 PAX8 0.52 1.69 1.21 2.36 0.0022 214457_at NM_006735 Hs.592177 HOXA2 0.72 2.06 1.40 3.03 0.0002 214608_s_at AJ000098 Hs.491997 EYA1 0.55 1.73 1.24 2.42 0.0013 214665_s_at AK000095 Hs.406234 CHP −0.52 0.59 0.43 0.82 0.0014 214822_at AF131833 Hs.495918 FAM5B 0.54 1.72 1.23 2.41 0.0017 215102_at AK026768 Hs.633705 DPY19L1P1 0.49 1.64 1.22 2.20 0.0011 215180_at AL109703 Hs.651358 — 0.43 1.54 1.16 2.06 0.0029 215289_at BE892698 — ZNF749 0.46 1.58 1.19 2.09 0.0017 215356_at AK023134 Hs.646351 ECAT8 0.46 1.58 1.15 2.17 0.0048 215476_at AF052103 Hs.159157 — 0.49 1.63 1.21 2.21 0.0016 215705_at BC000750 — PPP5C 0.52 1.68 1.22 2.32 0.0016 215715_at BC000563 Hs.78036 SLC6A2 0.75 2.12 1.37 3.29 0.0008 215850_s_at AK022209 Hs.651219 NDUFA5 0.48 1.62 1.18 2.23 0.0030 215944_at U80773 — — 0.49 1.64 1.20 2.24 0.0019 215953_at AL050020 Hs.127384 DKFZP564C196 0.47 1.59 1.16 2.19 0.0038 215973_at AF036973 — HCG4P6 0.55 1.74 1.30 2.32 0.0002 216050_at AK024584 Hs.406847 — 0.44 1.55 1.15 2.08 0.0035 216066_at AK024328 Hs.429294 ABCA1 0.50 1.65 1.22 2.22 0.0010 216240_at M34428 Hs.133107 PVT1 0.46 1.58 1.15 2.18 0.0046 216881_x_at X07882 Hs.528651 PRB4 0.41 1.51 1.14 1.99 0.0042 216989_at L13779 Hs.121494 SPAM1 0.46 1.58 1.15 2.16 0.0044 217004_s_at X13230 Hs.387262 MCF2 0.39 1.48 1.14 1.91 0.0032 217253_at L37198 Hs.632861 — 0.51 1.66 1.17 2.35 0.0041 217995_at NM_021199 Hs.511251 SQRDL −0.82 0.44 0.29 0.66 0.0001 218768_at NM_020401 Hs.524574 NUP107 0.63 1.88 1.31 2.70 0.0006 218881_s_at NM_024530 Hs.220971 FOSL2 −0.52 0.60 0.42 0.85 0.0044 218980_at NM_025135 Hs.436636 FHOD3 0.63 1.88 1.29 2.74 0.0011 219000_s_at NM_024094 Hs.315167 DCC1 1.06 2.90 1.89 4.44 0.0000 219171_s_at NM_007345 Hs.189826 ZNF236 0.56 1.76 1.20 2.56 0.0035 219182_at NM_024533 Hs.156784 FLJ22167 0.48 1.62 1.18 2.22 0.0027 219425_at NM_014351 Hs.189810 SULT4A1 0.74 2.11 1.41 3.14 0.0003 219520_s_at NM_018458 Hs.527524 WWC3 −0.49 0.61 0.44 0.84 0.0029 219537_x_at NM_016941 Hs.127792 DLL3 0.55 1.73 1.23 2.44 0.0018 219617_at NM_024766 Hs.468349 C2orf34 0.53 1.70 1.19 2.43 0.0035 219643_at NM_018557 Hs.470117 LRP1B 0.55 1.73 1.30 2.30 0.0001 219704_at NM_015982 Hs.567494 YBX2 0.75 2.12 1.42 3.16 0.0002 219882_at NM_024686 Hs.445826 TTLL7 0.51 1.66 1.18 2.35 0.0038 219937_at NM_013381 Hs.199814 TRHDE 0.54 1.71 1.23 2.38 0.0015 219955_at NM_019079 Hs.562195 L1TD1 0.60 1.82 1.25 2.65 0.0018 220029_at NM_017770 Hs.408557 ELOVL2 0.52 1.68 1.18 2.40 0.0038 220076_at NM_019847 Hs.156727 ANKH 0.77 2.17 1.53 3.07 0.0000 220294_at NM_014379 Hs.13285 KCNV1 0.45 1.56 1.16 2.11 0.0036 220366_at NM_022142 Hs.104894 ELSPBP1 0.53 1.69 1.19 2.41 0.0034 220394_at NM_019851 Hs.199905 FGF20 0.61 1.84 1.30 2.60 0.0006 220397_at NM_020128 Hs.591036 MDM1 0.41 1.51 1.17 1.95 0.0015 220541_at NM_021801 Hs.204732 MMP26 0.50 1.64 1.24 2.18 0.0006 220653_at NM_015363 — ZIM2 0.60 1.83 1.33 2.53 0.0002 220700_at NM_018543 Hs.188495 WDR37 0.59 1.80 1.22 2.66 0.0029 220703_at NM_018470 Hs.644603 C10orf110 0.59 1.80 1.26 2.58 0.0012 220771_at NM_016181 Hs.633593 LOC51152 0.60 1.81 1.23 2.67 0.0025 220817_at NM_016179 Hs.262960 TRPC4 0.47 1.60 1.19 2.14 0.0019 220834_at NM_017716 Hs.272789 MS4A12 0.52 1.68 1.27 2.22 0.0003 220847_x_at NM_013359 Hs.631598 ZNF221 0.50 1.65 1.19 2.28 0.0025 220852_at NM_014099 Hs.621386 PRO1768 0.48 1.62 1.19 2.20 0.0022 220970_s_at NM_030977 Hs.406714 KRTAP2-4/ 0.49 1.64 1.16 2.31 0.0050 LOC644350 220981_x_at NM_022053 Hs.648337 NXF2 0.45 1.56 1.19 2.05 0.0014 220993_s_at NM_030784 Hs.632612 GPR63 0.38 1.46 1.13 1.88 0.0041 221018_s_at NM_031278 Hs.333132 TDRD1 0.81 2.25 1.51 3.37 0.0001 221077_at NM_018076 Hs.127530 ARMC4 0.56 1.76 1.25 2.47 0.0013 221137_at AF118071 — — 0.46 1.59 1.15 2.20 0.0049 221168_at NM_021620 Hs.287386 PRDM13 0.68 1.96 1.33 2.91 0.0007 221258_s_at NM_031217 Hs.301052 KIF18A 0.62 1.86 1.34 2.58 0.0002 221319_at NM_019120 Hs.287793 PCDHB8 0.40 1.49 1.14 1.96 0.0041 221393_at NM_014627 — TAAR3 0.50 1.64 1.17 2.31 0.0043 221591_s_at BC005004 Hs.592116 FAM64A 0.72 2.05 1.38 3.05 0.0004 221609_s_at AY009401 Hs.29764 WNT6 0.40 1.50 1.15 1.95 0.0028 221718_s_at M90360 Hs.459211 AKAP13 −0.64 0.53 0.36 0.78 0.0013 221950_at AI478455 Hs.202095 EMX2 0.67 1.96 1.41 2.72 0.0001

TABLE 4 Features of 13 probe sets in the gene signature Rank of Rank of Rank of Entrez expression variation significant Gene Gene [n = 19619 [n = 19619 [n = 172 Probe Set Symbol Gene Title ID Coef.* (%)] (%)] (%)] 201243_s_at ATP1B1 ATPase, Na+/K+ transporting, beta 1 481 −0.54  517 (2.6) 2224 (11.3) 111 (64.5) polypeptide 203147_s_at TRIM14 Tripartite motif-containing 14 8518 −0.56  3532 (18.0) 9499 (48.4) 112 (65.1) 221591_s_at FAM64A Family with sequence similarity 64, member A 7372 0.72  6171 (31.5) 6108 (31.1)  29 (16.9) 218881_s_at FOSL2 FOS-like antigen 2 10614 −0.52  6526 (33.3) 12445 (63.4)  155 (90.1) 202814_s_at HEXIM1 Hexamethylene bis-acetamide inducible 1 11075 0.59  7415 (37.8) 9026 (46.0) 161 (93.6) 204179_at MB Myoglobin 9830 0.47  7703 (39.3) 7942 (40.5) 156 (90.7) 204584_at L1CAM L1 cell adhesion molecule 4151 0.56  9327 (47.5) 3329 (17.0) 17 (9.9) 202707_at UMPS Uridine monophosphate synthetase 3897 0.60 12311 (62.8) 18737 (95.5)  101 (58.7) 208399_s_at EDN3 Endothelin 3 4193 0.48 16344 (83.3) 8234 (42.0) 110 (64.0) 203001_s_at STMN2 Stathmin-like 2 2315 0.55 16948 (86.4) 5690 (29.0) 109 (63.4) 202490_at IKBKAP Inhibitor of kappa light polypeptide gene 23040 0.42 18769 (95.7) 10412 (53.1)   84 (48.8) enhancer in B-cells, kinase complex- associated protein 205386_s_at MDM2 Mdm2, transformed 3T3 cell double minute 2 7776 0.49 19251 (98.1) 14275 (72.8)  104 (60.5) 219171_s_at ZNF236 Zinc finger protein 236 54478 0.56 19383 (98.8) 17046 (86.9)  132 (76.7) *Coefficient of the Cox model

TABLE 4b Features of the 2 probe sets that were removed from the gene signature Rank of Rank of Rank of Entrez expression variation significant Gene Gene [n = 19619 [n = 19619 [n = 172 Probe Set Symbol Gene Title ID Coef.* (%)] (%)] (%)] 210016_at MYT1L Myelin transcription factor 1-like 1908 0.60 17902 (91.2) 18637 (95.0) 27 (15.7) 206426_at MLANA Melan-A 2355 0.63 19159 (97.7) 17172 (87.5) 81 (47.1) *Coefficient of the Cox model

TABLE 5 Demographic distributions of patients in validation sets Clinical DCC, All DCC, UM DCC, HLM DCC, MSK Duke UM-SQ Factors n = 360 (%) n = 177 (%) n = 79 (%) n = 104 (%) n = 89 (%) n = 129 (%) Pathology Type Adeno 360 (100) 177 (100)  79 (100) 104 (100) 43 (48) 0 Non-Adeno 0 (0) 0 (0) 0 (0) 0 (0) 46 (52) 129 (100) Disease stage I 220 (61)  116 (66)  41 (52) 63 (61) 67 (75) 73 (57) II 69 (19) 29 (16) 20 (25) 20 (19) 18 (20) 33 (25) III 69 (19) 32 (18) 16 (20) 21 (20) 3 (3) 23 (18) IV 0 (0) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) Unknown 2 (1) 0 (0) 2 (3) 0 (0) 0 (0) 0 (0) Adjuvant chemotherapy No 210 (58)  76 (43) 61 (77) 73 (70)  89 (100) NS Yes 64 (18) 17 (10) 16 (20) 31 (30) 0 (0) NS Unknown 86 (24) 84 (47) 2 (3) 0 (0) 0 (0) NS Adjuvant radiotherapy No 209 (58)  76 (43) 57 (72) 76 (73)  89 (100) NS Yes 64 (18) 17 (10) 19 (24) 28 (27) 0 (0) NS Unknown 87 (24) 84 (47) 3 (4) 0 (0) 0 (0) NS Age (year)  <65 163 (45)  87 (49) 17 (34) 49 (47) 33 (37) 52 (40) ≧65 197 (55) 90 (51) 25 (66) 55 (53) 56 (63) 77 (60) Gender Male 177 (49)  100 (56)  40 (51) 37 (36) 54 (61) 82 (64) Female 183 (51)  77 (44) 39 (49) 67 (64) 35 (39) 47 (36) DCC: Directors' Challenge Consortium; UM: University of Michigan; HLM: H. Lee Moffitt Cancer Center; MSK: Memorial Sloan-Kettering Cancer Center; NS: Not specified

TABLE 6 Adjuvant therapies in the Director's Challenge Consortium (DCC) Patients Adjuvant radiotherapy Adjuvant Chemotherapy No Yes Unknown Total All No 190 20 0 210 Yes 19 44 1 64 Unknown 0 0 86 86 University of Michigan (UM) No 76 0 0 76 Yes 0 17 0 17 Unknown 0 0 84 84 H. Lee Moffitt (HLM) No 51 10 0 61 Yes 6 9 1 16 Unknown 0 0 2 2 Memorial Sloan-Kettering (MSK) No 63 10 0 73 Yes 13 18 0 31 Unknown 0 0 0 0

TABLE 7 Primers for qPCR validation SEQ SEQ ID Amplicon ID Amplicon Gene NO Forward Length Tm NO Reverse Length Tm FAM64A 173 TCCTCAACAGAGCCCCTC 18 62.6 186 TTCTGGAGATGCTGAATTCCC 21 62 MB 174 AGTTGGTGCTGAACGTCTG 19 62.2 187 GGTGACCCTTAAAGAGCCTG 20 62 EDN3 175 GTGTCTACTATTGCCACCTGG 21 62 188 GCTTCCTCTGTAGTTGGACAG 21 61.9 ZNF236 176 ACGTAGACCAGTTTGAAGAGC 21 62 189 GGCTGCTGATGGTACGTATC 20 62 FOSL2 177 ACGCCGAGTCCTACTCCA 18 59 190 TGAGCCAGGCATATCTACCC 20 60 L1CAM 178 GCCCCCCAGGTTCAGTACCGCG 22 59 191 CTGACAATCTGCTCCTGCCA 20 60 TRIM14 179 CCTCAGCATCCTGAATACATCA 22 60 192 TGTGTCAGGTTCCTGTGCTG 20 60 STMN2 180 GAACCTCGCAACATCAACATC 21 61.7 193 GGCTTCAAGATCAGCTCAAAAG 22 61.8 UMPS 181 ACACAGTGAAAAAGCAGTATGAAG 24 61.9 194 TTTCACAACTCCTGAGCCTG 20 62.1 ATP1B1 182 CCAAATGTCCTTCCCGTTCAG 21 63 195 TGCCCAGTCCAAAATACTCC 20 62 HEXIM1 183 TCGAGGACTCTACTAGCCATG 21 62.1 196 GCTCTTCCTGGACAGCAG 18 61.7 IKBKAP 184 GGAAGCAAGCCCTCTGTGT 19 60 197 TCTGCTCAACCAGCTTTCCT 20 60 MDM2 185 CATGTCTGTACCTACTGATGGTG 23 62 198 GCAATGGCTTTGGTCTAACC 20 61.5

TABLE 8 Risk group based on 15-gene signature in stage I patients n HR 95% CI p value BR.10 34 13.3  2.9-62.1 <0.0001 Observation arm DCC 141 3.3 1.5-7.4 0.002 No adjuvant therapy UM 57 1.9 0.6-6.1 0.28 HLM 37 2.5 0.9-6.9 0.07 MSK 47 NA NA 0.05 Duke 67 1.06 0.5-2.2 0.88 UM-SQ 73 1.4 0.6-3.1 0.44 n: number of patients; HR: hazard ratio; CI: confidence interval * HR and CI cannot be calculated as no death occurred in the good prognosis group, p value the score test.

TABLE 9 Probe set target sequences of the 13-gene signature SEQ ID Probe NO: set ID Target sequence 35 205386_S_AT tttcccctagttgacctgtctataagagaattatatatttctaactatataaccctaggaattt agacaacctgaaatttattcacatatatcaaagtgagaaaatgcctcaattcacatagat ttcttctctttagtataattgacctactttggtagtggaatagtgaatacttactataatttgactt gaatatgtagctcatcctttacaccaactcctaattttaaataatttctactctgtcttaaatga gaagtacttggttttttttttcttaaatatgtatatgacatttaaatgtaacttattattttttttgaga ccgagtcttgctctgttacccaggctggagtgcagtgggtgatcttggctcactgcaagct ctgccctccccgggttcgcaccattctcctgcctcagcctcccaattagcttggcctacag tcatctgcc 78 208399_S_AT ccgagccgagcttactgtgagtgtggagatgttatcccaccatgtaaagtcgcctgcgc aggggagggctgcccatctccccaacccagtcacagagagataggaaacggcattt gagtgggtgtccagggccccgtagagagacatttaagatggtgtatgacagagcattg gccttgaccaaatgttaaatcctctgtgtgtatttcataagttattacaggtataaaagtgat gacctatcatgaggaaatgaaagtggctgatttgctggtaggattttgtacagtttagaga agcgattatttattgtgaaactgttctccactccaactcctttatgtggatctgttcaaagtagt cactgtatatacgtatagagaggtagataggtaggtagattttaaattgcattctgaatac aaactcatactccttagagcttgaattacatttttaaaatgcatatgtgctgtttggcaccgt ggcaagatggtatcagagagaaacccatcaattgctcaaatactc 4 201243_S_AT ggtgatgggttgtgttatgcttgtattgaatgctgtcttgacatctcttgccttgtcctccggtat gttctaaagctgtgtctgagatctggatctgcccatcactttggcctagggacagggctaa ttaatttgctttatacattttcttttactttccttttttcctttctggaggcatcacatgctggtgctgt gtctttatgaatgttttaaccattttcatggtggaagaattttatatttatgcagttgtacaatttt atttttttctgcaagaaaaagtgtaatgtatgaaataaaccaaagtcacttgtttgaaaata aatctttattttgaactttataaaagcaatgcagtaccccatagactggtgttaaatgttgtct acagtgcaaaatccatgttctaacatatgtaataattgccaggagtacagtgctcttgttg atcttgtattcagtcaggttaaaa 22 204179_AT tgttccggaaggacatggcctccaactacaaggagctgggcttccagggctaggcccc tgccgctcccacccccacccatctgggccccgggttcaagagagagcggggtctgatc tcgtgtagccatatagagtttgcttctgagtgtctgctttgtttagtagaggtgggcaggagg agctgaggggctggggctggggtgttgaagttggctttgcatgcccagcgatgcgcctc cctgtgggatgtcatcaccctgggaaccgggagtgcccttggctcactgtgttctgcatg gtttggatctgaattaattgtcctttcttctaaatcccaaccgaacttcttccaacctccaaac tggctgtaaccccaaatccaagccattaactacacctgacagtagcaattgtctgattaa tcactggccccttgaagacagcagaatgtccctttgcaatgaggaggagatctgggctg ggcgggccagctggggaagcatttgactatctggaacttgtgtgtgcctcctcaggtatg gca 169 221591_S_AT cacatctggacccatcagtgactgcctgccatagcctgagagtgtcttggggagacctt gcagagggggagaattgttccttctgctttcctaggggactcttgagcttagaaactcatc gtacacttgaccttgagccttctatttgcctcatctataacatgaagtgctagcatcagatat ttgagagctcttagctctgtacccgggtgcctggtttttggggagtcatccgcagagtcact cacccactgtgtttctggtgccaaggctcttgagggccccactctcatccctcctttcccta ccagggactcggaggaaggcataggagatatttccaggcttacgaccctgggctcac gggtacctatttatatgctcagtgcagagcactgtggatgtgccaggaggggtagccct gttcaagagcaatttctgccctttgtaaattatttaagaaacctgctttgtcattttattagaaa gaaaccagcgtgtgactttcctagataacactgctttc 15 203147_S_AT accaatcacgcctacagtgctttgaaggtttcctctcctaggctagtttcaaacaggccct aaacaagtctgctgctgccctctcatcagacctccgcaccctcaccccaccatcactta nactactttaatccagttccttcaaagtgatacccccacaggtaagccctcagcatcctg aatacatcatccgcagcctgggaaccttctccctcgtacagcacaggaacctgacaca tagtaggcacacagtaaacgtttgtgaatgaatgggagtcatccagtcctgactcttctgt ctcttgaggtcccttgaatcttccgcttcctccccaccgatttcagcgtgtccacatcacag ctccctccagaagctgcaagagcttcttagcagttcctggtctgaaccctctcccagtcct catcttccaccctaaaactagagtgatcttcctaaaacttcacttaacccctcagctatga aaaggcttccaggagtttccatgaa 130 218881_S_AT aggtcacagtatcctcgtttgaaagataattaagatcccccgtggagaaagcagtgac acattcacacagctgttccctcgcatgttatttcatgaacatgacctgttttcgtgcactaga cacacagagtggaacagccgtatgcttaaagtacatgggccagtgggactggaagtg acctgtacaagtgatgcagaaaggagggtttcaaagaaaaaggattttgtttaaaatac tttaaaaatgttatttcctgcatcccttggctgtgatgcccctctcccgatttcccaggggctc tgggagggacccttctaagaagattgggcagttgggtttctggcttgagatgaatccaag cagcagaatgagccaggagtagcaggagatgggcaaagaaaactggggtgcactc agctctcacaggggtaatca 11 202490_AT gaggatggcacaagcgattcacgtaggatctgcccctgtgaccaaaacacctcccatt gggccccacttccaacactggtgatcacatttcaacatgaggtttagggaaacaaatgc ctaaactacagcactgtacataaactaacaggaaatgctgcttttgatcctcaaagaagt gatatagccaaaattgtaatttaagaagcctttgtcagtatagcaagatgttaactataga atcaatctaggagtattcactgtaaaattcaacttttctgtatgtttgaacattttcacaatctc ataggagtttttaaaaagaagagaaagaagatatactttgctttggagaaatctactttttg acttacatgggtttgctgtaattaagtgcccaatattgaaaggctgcaagtactttgtaatc actctttggcatgggtaaataagcatggtaacttatattgaaatatagtgctcttgctttggat aactgtaaagggacccatgctgatagactggaaa 12 202707_AT aagttcattcttaagcttgctttttttgagactggtgtttgttagacagccacagtcctgtctgg gttagggtcttccacatttgaggatccttcctatctctccatgggactagactgctttgttattc tatttattttttaatttttttcgagacaggatctcactctgttgcccaggatggagtgcagtggt gagatcacggctcattgcagcctcgacctcccaggtgatcctcccacctcagcttccag attagctggtgctataggcatgcaccaccacgtccatctaaatttctttattatttgtagagat gaggtcttgccatgttacccaggctggtctcaactcctgggctcaagcgatcctcctgcct cagtctctcaaagtgctgggattacaggtgtgagccactgtgcccagcctaattgcagta agacaa 14 203001_S_AT acctcgcaacatcaacatctatacttacgatgatatggaagtgaagcaaatcaacaaa cgtgcctctggccaggcttttgagctgatcttgaagccaccatctcctatctcagaagccc cacgaactttagcttctccaaagaagaaagacctgtccctggaggagatccagaaga aactggaggctgcaggggaaagaagaaagtctcaggaggcccaggtgctgaaaca attggcagagaagagggaacacgagcgagaagtccttcagaaggctttggaggaga acaacaacttcagcaagatggcggaggaaaagctgatcctgaaaatggaacaaatt aaggaaaaccgtgaggctaatctagctgctattattgaacgtctgcaggaaaaggaga ggcatgctgcggaggtgcgcaggaacaaggaactccaggttgaactgtctggctgaa gcaagggagggtctggcacgcc 13 202814_S_AT tgcctctcgcgcatggaggacgagaacaaccggctgcggctggagagcaagcggct gggtggcgacgacgcgcgtgtgcgggagctggagctggagctggaccggctgcgcg ccgagaacctccagctgctgaccgagaacgaactgcaccggcagcaggagcgagc gccgctttccaagtttggagactagactgaaacttttttgggggagggggcaaagggga ctttttacagtgatggaatgtaacattatatacatgtgtatataagacagtggacctttttatg acacataatcagaagagaaatccccctggctttggttggtttcgtaaatttagctatatgta gcttgcgtgctttctcctgttcttttaattatgtgaaactgaagagttgcttttcttgttttccttttta gaagtttttttccttaatgtgaaagtaatttgaccaagttataatgcatttttgtttttaacaaat cccctccttaaacggagctataaggtggccaaatctga 133 219171_S_AT cttttgttcttgctgggttatttattttgattttagcattaaatgtcatctcaggatatctctaaaag gggttgtttaattcctaattgtatagaaagctagtttggtgaattgtattggttaattgactgttt aaggccttaacaggtgaatctagagcctacttttattttggttaaagaaaaagaaaatatc aataattcaattttgtgtcttttctcaatttattagcaaacacaagacattttatgtattatttcga tttacttcctaattataaaagctgcttttttgcagaacattccttgaaaatataaggttttgaaa agacataattttacttgaatctttgtggggtacaggttgatctttatattttactggttgttttaaa aattctagaaaagagatttctaggcctcatgtataaccagggttttgaggataaagaact gtatttttagaactatctcatcatagcatatctgctttggaataactat 26 204584_AT cctccctatcgtctgaacagttgtcttcctcagcctcctcccgcccccaccttgggaatgta aatacaccgtgactttgaaagtttgtacccctgtccttccctttacgccactagtgtgtaggc agatgtctgagtccctaggtggtttctaggattgatagcaattagctttgatgaacccatcc caggaaaaataaaaacagacaaaaaaaaaggaaagattggttctcccagcactgct cagcagccacagcctccctgtatgcctgtgcttggtctactgataagccctctacaaaa

TABLE 10 Coefficient of individual genes in 13-gene signature: Principal Component values Gene Gene Symbol Probe set pc1 pc2 pc3 pc4 1 ATP1B1 201243_s_at −0.189 −0.423 0.229 0.059 2 IKBKAP 202490_at 0.364 0.070 −0.357 −0.120 3 UMPS 202707_at 0.353 −0.009 0.136 0.011 4 HEXIM1 202814_s_at −0.108 0.504 0.265 0.279 5 STMN2 203001_s_at 0.326 0.044 −0.100 −0.122 6 TRIM14 203147_s_at −0.148 0.212 0.132 −0.368 7 MB 204179_at 0.197 0.028 0.548 −0.161 8 L1CAM 204584_at 0.042 0.510 0.077 0.276 9 MDM2 205386_s_at 0.180 0.081 0.325 −0.500 10 EDN3 208399_s_at 0.413 0.042 −0.188 −0.260 11 FOSL2 218881_s_at 0.036 −0.209 −0.225 0.190 12 ZNF236 219171_s_at 0.188 −0.313 0.297 0.332 13 FAM64A 221591_s_at 0.283 0.216 −0.174 0.320 Eigenvalues of principal 3.33 1.82 1.37 1.32 components Weight of each PC for risk 0.557 0.328 0.430 0.335 score

Risk score=0.557*PC1+0.328*PC2+0.43*PC3+0.335*PC4

where

PC1=Sum [pc1*(expression data)]_(Gene 1-13)

PC2=Sum [pc2*(expression data)]_(Gene 1-13)

PC3=Sum [pc3*(expression data)]_(Gene 1-13)

PC4=Sum [pc4*(expression data)]_(Gene 1-13)

Patients classified as high risk or lower risk according to risk score≧−0.1 or <−0.1.

TABLE 11 Probe set target sequences for 172 genes SEQ ID Probe Gene NO: Set ID Symbol Target Sequence 1 200878_at EPAS1 cactttgcaactccctgggtaagagggacgacacctctggtttttcaataccaattac atggaacttttctgtaatgggtacnaatgaagaagtttctaaaaacacacacaaagc acattgggccaactatttagtaagcccggatagacttattgccaaaaacaaaaaata gctttcaaaagaaatttaagttctatgagaaattccttagtcatggtgttgcgtaaatc atattttagctgcacggcattaccccacacagggtggcagaacttgaagggttactg acgtgtaaatgctggtatttgatttcctgtgtgtgttgccctggcattaagggcatttta cccttgcagttttactaaaacactgaaaaatattccaagcttcatattaaccctacctg tcaacgtaacgat 2 201228_s_at ARIH2 cctacccacctcaaaatgtctgtactgcaagagggccctgggcctctgctttccatatt cacgtttggccagagttgtagtcccaaagaagagcatgggtggcagatggtaggga attgaactggcctgtgcaatgggcatggagcacaaggggtcacagcatgcctcctgc cttaccgtggcagtacggagacagtccagaacatggtcttcttgccacggggtgttgt tgtctctggtggtgctgcatgtctgtggctcacctttattcttgaaactgaggtttacct ggatctggctactgaggctagagcccacagcagaatggggttgggcctgtggccccc caaactagggggtgtgggttcatcacagtgttgccttttgtctcctaaagatagggat ctacttttgaagggaattgttcctcccaaata 3 201242_s_at ATP1B1 agagctgatcacaagcacaaatctttcccactagccatttaataagttaaaaaaaga tacaaaaacaaaaacctactagtcttgaacaaactgtcatacgtatgggacctacac ttaatctatatgctttacactagctttctgcatttaataggttagaa 4 201243_s_at ATP1B1 ggtgatgggttgtgttatgcttgtattgaatgctgtcttgacatctcttgccttgtcctcc ggtatgttctaaagctgtgtctgagatctggatctgcccatcactttggcctagggaca gggctaattaatttgctttatacattttcttttactttccttttttcctttctggaggcatca catgctggtgctgtgtctttatgaatgttttaaccattttcatggtggaagaattttatat ttatgcagttgtacaattttatttttttctgcaagaaaaagtgtaatgtatgaaataaa ccaaagtcacttgtttgaaaataaatctttattttgaactttataaaagcaatgcagta ccccatagactggtgttaaatgttgtctacagtgcaaaatccatgttctaacatatgta ataattgccaggagtacagtgctcttgttgatcttgtattcagtcaggttaaaa 5 201301_s_at ANXA4 ggtgaaatttctaactgttctctgttcccggaaccgaaatcacctgttgcatgtgtttg atgaatacaaaaggatatcacagaaggatattgaacagagtattaaatctgaaaca tctggtagctttgaagatgctctgctggctatagtaaagtgcatgaggaacaaatctg catattttgctgaaaagctctataaatcgatgaagggcttgggcaccgatgataaca ccctcatcagagtgatggtttctcgagcagaaattgacatgttggatatccgggcaca cttcaagagactctatggaaagtctctgtactcgttcatcaagggtgacacatctgga gactacaggaaagtactgcttgttctctgtggaggagatgattaaaataaaaatccc agaaggacaggaggattctcaacactttgaatttttttaacttcatttttctacactgct attatcattatctc 6 201502_s_at NFKBIA ccaactacaatggccacacgtgtctacacttagcctctatccatggctacctgggcat cgtggagcttttggtgtccttgggtgctgatgtcaatgctcaggagccctgtaatggcc ggactgcccttcacctcgcagtggacctgcaaaatcctgacctggtgtcactcctgtt gaagtgtggggctgatgtcaacagagttacctaccagggctattctccctaccagctc acctggggccgcccaagcacccggatacagcagcagctgggccagctgacactaga aaaccttcagatgctgccagagagtgaggatgaggagagctatgacacagagtcag agttcacggagttcacagaggacgagctgccctatgatgactgtgtgtttggaggcc agcgtctgacgttatgag 7 202023_at EFNA1 ccaccttcacctcggagggacggagaaagaagtggagacagtcctttcccaccattc ctgcctttaagccaaagaaacaagctgtgcaggcatggtcccttaaggcacagtggg agctgagctggaaggggccacgtggatgggcaaagcttgtcaaagatgccccctcc aggagagagccaggatgcccagatgaactgactgaaggaaaagcaagaaacagtt tcttgcttggaagccaggtacaggagaggcagcatgcttgggctgacccagcatctc ccagcaagacctcatctgtggagctgccacagagaagtttgtagccaggtactgcat tctctcccatcctggggcagcactccccagagctgtgccagcaggggggctgtgcca acctgttcttagagtgtagctgtaagggcagtgcccatgtgtacattctgcctagagtg tagcctaaagggcagggcccacgtgtatagtatctgta 8 202035_s_at SFRP1 tcggccagcgagtacgactacgtgagcttccagtcggacatcggcccgtaccagagc gggcgcttctacaccaagccacctcagtgcgtggacatccccgcggacctgcggctg tgccacaacgtgggctacaagaagatggtgctgcccaacctgctggagcacgagac catggcggaggtgaagcagcaggccagcagctgggtgcccctgctcaacaagaact gccacgccggcacccaggtcttcctctgctcgctcttcgcgcccgtctgcctggaccg gcccatctacccgtgtcgctggctctgcgaggccgtgcgcgactcgtgcgagccggtc atgcagttcttcggcttctactggcccgagatgcttaagtgtgacaagttccccgagg gggacgtctgcatcgccatgacgccgcccaatgccaccgaagcctccaagccccaa ggcacaacggtgtgtcctccctgtgacaacgagttgaaatctgaggccatcattgaa catctctgt 9 202036_s_at SFRP1 gacaaaccatttccaacagcaacacagccactaaaacacaaaaagggggattggg cggaaagtgagagccagcagcaaaaactacattttgcaacttgttggtgtggatcta ttggctgatctatgcctttcaactagaaaattctaatgattggcaagtcacgttgttttc aggtccagagtagtttctttctgtctgctttaaatggaaacagactcataccacactta caattaaggtcaagcccagaaagtgataagtgcagggaggaaaagtgcaagtcca ttatgtaatagtgacagcaaaggcccaggggagaggcattgccttctctgcccacag tctttccgtgtgattgtctttgaatctgaatcagccagtctcagatgccccaaagtttcg gttcctatgagcccggggcatgatctgatccccaagacatg 10 202037_s_at SFRP1 taacacttggctcttggtacctgtgggttagcatcaagttctccccagggtagaattca atcagagctccagtttgcatttggatgtgtaaattacagtaatcccatttcccaaacct aaaatctgtttttctcatcagactctgagtaactggttgctgtgtcataacttcatagat gcaggaggctcaggtgatctgtttgaggagagcaccctaggcagcctgcagggaat aacatactggccgttctgacctgttgccagcagatacacaggacatggatgaaattc ccgtttcctctagtttcttcctgtagtactcctcttttagatcc 11 202490_at IKBKAP gaggatggcacaagcgattcacgtaggatctgcccctgtgaccaaaacacctcccat tgggccccacttccaacactggtgatcacatttcaacatgaggtttagggaaacaaa tgcctaaactacagcactgtacataaactaacaggaaatgctgcttttgatcctcaaa gaagtgatatagccaaaattgtaatttaagaagcctttgtcagtatagcaagatgtta actatagaatcaatctaggagtattcactgtaaaattcaacttttctgtatgtttgaac attttcacaatctcataggagtttttaaaaagaagagaaagaagatatactttgcttt ggagaaatctactttttgacttacatgggtttgctgtaattaagtgcccaatattgaaa ggctgcaagtactttgtaatcactctttggcatgggtaaataagcatggtaacttatat tgaaatatagtgctcttgctttggataactgtaaagggacccatgctgatagactgga aa 12 202707_at UMPS aagttcattcttaagcttgctttttttgagactggtgtttgttagacagccacagtcctg tctgggttagggtcttccacatttgaggatccttcctatctctccatgggactagactgc tttgttattctatttattttttaatttttttcgagacaggatctcactctgttgcccaggat ggagtgcagtggtgagatcacggctcattgcagcctcgacctcccaggtgatcctccc acctcagcttccagattagctggtgctataggcatgcaccaccacgtccatctaaatt tctttattatttgtagagatgaggtcttgccatgttacccaggctggtctcaactcctgg gctcaagcgatcctcctgcctcagtctctcaaagtgctgggattacaggtgtgagcca ctgtgcccagcctaattgcagtaagacaa 13 202814_s_at HEXIM1 tgcctctcgcgcatggaggacgagaacaaccggctgcggctggagagcaagcggct gggtggcgacgacgcgcgtgtgcgggagctggagctggagctggaccggctgcgcg ccgagaacctccagctgctgaccgagaacgaactgcaccggcagcaggagcgagc gccgctttccaagtttggagactagactgaaacttttttgggggagggggcaaaggg gactttttacagtgatggaatgtaacattatatacatgtgtatataagacagtggacc tttttatgacacataatcagaagagaaatccccctggctttggttggtttcgtaaattt agctatatgtagcttgcgtgctttctcctgttcttttaattatgtgaaactgaagagttg cttttcttgttttcctttttagaagtttttttccttaatgtgaaagtaatttgaccaagtta taatgcatttttgtttttaacaaatcccctccttaaacggagctataaggtggccaaat ctga 14 203001_s_at STMN2 acctcgcaacatcaacatctatacttacgatgatatggaagtgaagcaaatcaacaa acgtgcctctggccaggcttttgagctgatcttgaagccaccatctcctatctcagaag ccccacgaactttagcttctccaaagaagaaagacctgtccctggaggagatccaga agaaactggaggctgcaggggaaagaagaaagtctcaggaggcccaggtgctgaa acaattggcagagaagagggaacacgagcgagaagtccttcagaaggctttggag gagaacaacaacttcagcaagatggcggaggaaaagctgatcctgaaaatggaac aaattaaggaaaaccgtgaggctaatctagctgctattattgaacgtctgcaggaaa aggagaggcatgctgcggaggtgcgcaggaacaaggaactccaggttgaactgtct ggctgaagcaagggagggtctggcacgcc 15 203147_s_at TRIM14 accaatcacgcctacagtgctttgaaggtttcctctcctaggctagtttcaaacaggcc ctaaacaagtctgctgctgccctctcatcagacctccgcaccctcaccccaccatcac ttanactactttaatccagttccttcaaagtgatacccccacaggtaagccctcagca tcctgaatacatcatccgcagcctgggaaccttctccctcgtacagcacaggaacctg acacatagtaggcacacagtaaacgtttgtgaatgaatgggagtcatccagtcctga ctcttctgtctcttgaggtcccttgaatcttccgcttcctccccaccgatttcagcgtgtc cacatcacagctccctccagaagctgcaagagcttcttagcagttcctggtctgaacc ctctcccagtcctcatcttccaccctaaaactagagtgatcttcctaaaacttcactta acccctcagctatgaaaaggcttccaggagtttccatgaa 16 203438_at STC2 gtccacattcctgcaagcattgattgagacatttgcacaatctaaaatgtaagcaaa gtagtcattaaaaatacaccctctacttgggctttatactgcatacaaatttactcatg agccttcctttgaggaaggatgtggatctccaaataaagatttagtgtttattttgagc tctgcatcttaacaagatgatctgaacacctctcctttgtatcaataaatagccctgtt attctgaagtgagaggaccaagtatagtaaaatgctgacatctaaaactaaataaat agaaaacaccaggccagaactatagtcatactcacacaaagggagaaatttaaact cgaaccaagcaaaaggcttcacggaaatagcatggaaaaacaatgcttccagtggc cacttcctaaggaggaacaaccccgtctgatctcagaattggcaccacgtgagcttg ctaagtgataatatctgtttctactacggatttaggcaacaggacctgtacattgtcac attgcat 17 203444_s_at MTA2 cacaaaggataccagggccctacggaaggctctgacccatctggaaatgcggcgag ctgctcgccgacccaacttgcccctgaaggtgaagccaacgctgattgcagtgcggc cccctgtccctctacctgcaccctcacatcctgccagcaccaatgagcctattgtcctg gaggactgagcacctgtggggaagggaggtgggctgagaggtagagggtggatgc ccagggcacccaaacctcccttccctttcgtgtcgaagggagtgaggagtgaattaa ggaagagagcaagtgagtgtgtgtccctggaggggttgggcgccctctggtgttacc acctcgagacttgtctcatgcctccatgcttgccgatggaggacagactgcaggaact tggcccatgtgggaacctagcctgttttggggggtaggacccacagatgtcttggac 18 203475_at CYP19A1 gaaattctttcccagtctgtcgatttatgcctcagccacttgcctgtgctacaattcatt gtgttacctgtagattcaggtaatacaaaccatatataatcatcaagtaatacaaact aatttagtaatagcctgggttaagtattattagggccctgtgtctgcatgtagaaaaa aaaattcacatgatgcacttcaaattcaaataaaaatccttttggcatgttcccattttt gcttagctcaattagtgtggctaaccaagagataactgtaaatgtgacattgatttgc tcttactacagctacagtgattgggggaggaaaagtcccaacccaatgggctcaaac ttctaaggggtactcctctcatccccttatccttctccctcgacattttctccctctttctt cccatgaccccaaagccaagggcaacagatcagtaaagaacgtggtcagagtaga acccctg 19 203509_at SORL1 gaatatcacagcttaccttgggaatactactgacaatttctttaaaatttccaacctga agatgggtcataattacacgttcaccgtccaagcaagatgcctttttggcaaccagat ctgtggggagcctgccatcctgctgtacgatgagctggggtctggtgcagatgcatct gcaacgcaggctgccagatctacggatgttgctgctgtggtggtgcccatcttattcct gatactgctgagcctgggggtggggtttgccatcctgtacacgaagcaccggaggct gcagagcagcttcaccgccttcgccaacagccactacagctccaggctggggtccgc aatcttctcctctggggatgacctgggggaagatgatgaagatgcccctatgataact ggattttcagatgacgtccccatggtgatagcctgaaagagctttcctcactagaaac ca 20 203928_x_at MAPT gagtccagtcgaagattgggtccctggacaatatcacccacgtccctggcggaggaa ataaaaagattgaaacccacaagctgaccttccgcgagaacgccaaagccaagac agaccacggggcggagatcgtgtacaagtcgccagtggtgtctggggacacgtctcc acggcatctcagcaatgtctcctccaccggcagcatcgacatggtagactcgcccca gctcgccacgctagctgacgaggtgtctgcctccctggccaagcagggtttgtgatca ggcccctggggcggtcaataatngtggagaggagagaatgagagagtgtggaaaa aaaaagaataatgacccggcccccgccctctgcccccagctgctcctcgcagttcgg ttaattggttaatcacttaacctgcttttgtcactc 21 203973_s_at CEBPD aagcggcgcaaccaggagatgcagcagaagttggtggagctgtcggctgagaacg agaagctgcaccagcgcgtggagcagctcacgcgggacctggccggcctccggcag ttcttcaagcagctgcccagcccgcccttcctgccggccgccgggacagcagactgc cggtaacgcgcggccggggcgggagagactcagcaacgacccatacctcagaccc gacggcccggagcggagcgcgccctgccctggcgcagccagagccgccgggtgccc gctgcagtttcttgggacataggagcgcaaagaagctacagcctggacttaccacca ctaaactgcgagagaagctaaacgtgtttattttcccttaaattatttttgtaatggta gctttttctacatcttactcctgttgatgcagctaaggtacatttgtaaaaagaaaaaa aaccagacttttcagacaaaccctttgtattgtagataagaggaaaagactgagcat gctcacttttttatattaa 22 204179_at MB tgttccggaaggacatggcctccaactacaaggagctgggcttccagggctaggccc ctgccgctcccacccccacccatctgggccccgggttcaagagagagcggggtctga tctcgtgtagccatatagagtttgcttctgagtgtctgctttgtttagtagaggtgggca ggaggagctgaggggctggggctggggtgttgaagttggctttgcatgcccagcgat gcgcctccctgtgggatgtcatcaccctgggaaccgggagtgcccttggctcactgtg ttctgcatggtttggatctgaattaattgtcctttcttctaaatcccaaccgaacttcttc caacctccaaactggctgtaaccccaaatccaagccattaactacacctgacagtag caattgtctgattaatcactggccccttgaagacagcagaatgtccctttgcaatgag gaggagatctgggctgggcgggccagctggggaagcatttgactatctggaacttgt gtgtgcctcctcaggtatggca 23 204267_x_at PKMYT1 ctgtggtgcatggcagcggaggccctgagccgagggtgggccctgtggcaggccct gcttgccctgctctgctggctctggcatgggctggctcaccctgccagctggctacag cccctgggcccgccagccaccccgcctggctcaccaccctgcagtttgctcctggaca gcagcctctccagcaactgggatgacgacagcctagggccttcactctcccctgagg ctgtcctggcccggactgtggggagcacctccaccccccggagcaggtgcacaccca gggatgccctggacctaagtgacatcaactcagagcctcctcggggctccttcccctc ctttgagcctcggaacctcctcagcctgtttgaggacaccctagacccaacctgagcc ccagactctgcctctgcacttttaaccttttatcctgtgtctctcccgtcgcccttgaaa gctggggcccctcgggaactcccatggtcttctctgcctggccgtgtctaataa 24 204338_s_at RGS4 gaaacatcggctaggtttcctgctgcaaaaatctgattcctgtgaacacaattcttccc acaacaagaaggacaaagtggttatttgccagagagtgagccaagaggaagtcaa gaaatgggctgaatcactggaaaacctgattagtcatgaatgtgggctggcagcttt caaagctttcttgaagtctgaatatagtgaggagaatattgacttctggatcagctgt gaagagtacaagaaaatcaaatcaccatctaaactaagtcccaaggccaaaaaga tctataatgaattcatctcagtccaggcaaccaaagaggtgaacctggattcttgcac cagggaagagacaagccggaacatgctagagcctacaataacctgctttgatgagg cccagaagaagattttcaacctgatggagaaggattcctaccgccgcttcctcaagtc tcgattctatcttgatttggtcaacccgtcca 25 204531_s_at BRCA1 ttcaagaaccggtttccaaagacagtcttctaattcctcattagtaataagtaaaatgt ttattgttgtagctctggtatataatccattcctcttaaaatataagacctctggcatga atatttcatatctataaaatgacagatcccaccaggaaggaagctgttgctttctttga ggtgatttttttcctttgctccctgttgctgaaaccatacagcttcataaataattttgct tgctgaaggaagaaaaagtgtttttcataaacccattatccaggactgtttatagctg ttggaaggactaggtcttccctagcccccccagtgtgcaagggcagtgaagacttga ttgtaca 26 204584_at L1CAM cctccctatcgtctgaacagttgtcttcctcagcctcctcccgcccccaccttgggaat gtaaatacaccgtgactttgaaagtttgtacccctgtccttccctttacgccactagtgt gtaggcagatgtctgagtccctaggtggtttctaggattgatagcaattagctttgatg aacccatcccaggaaaaataaaaacagacaaaaaaaaaggaaagattggttctcc cagcactgctcagcagccacagcctccctgtatgcctgtgcttggtctactgataagc cctctacaaaa 27 204684_at NPTX1 ttccttttgtagattcccagtttattttctaagactgcaaagatcactttgtcaccagcc ctgggacctgagaccaagggggtgtcttgtgggcagtgagggggtgaggagaggct ggcatgaggttcagtcattccagtgagctccaaagaggggccacctgttctcaaaag catgttggggaccaggaggtaaaactggccatttatggtgaacctgtgtcttggagct gacttactaagtggaatgagccgaggatttgaatatcagttctaaccttgatagaag aaccttgggttacatgtggttcacattaagaggatagaatcctttggaatcttatggc aaccaaatgtggcttgacgaagtcgtggtttcatctctt 28 204810_s_at CKM gcaagcaccccaagttcgaggagatcctcacccgcctgcgtctgcagaagaggggt acaggtgcggtggacacagctgccgtgggctcagtatttgacgtgtccaacgctgat cggctgggctcgtccgaagtagaacaggtgcagctggtggtggatggtgtgaagctc atggtggaaatggagaagaagttggagaaaggccagtccatcgacgacatgatccc cgcccagaagtaggcgcctgcccacctgccaccgactgctggaaccccagccagtg ggagggcctggcccaccagagtcctgctccctcactcctcgccccgccccctgtccca gagtccacctgggggctctctccacccttctcagagttccagtttcaaccagagttcca accaatgggctccatcctctggattctggccaatgaaatatctccctggcagggtcct cttcttttcccagagctcctccccaaccaggagctctagttaatg 29 204817_at ESPL1 tgtttggctgtagcagtgcggccctggctgtgcatggaaacctggagggggctggca tcgtgctcaagtacatcatggctggttgccccttgtttctgggtaatctctgggatgtga ctgaccgcgacattgaccgctacacggaagctctgctgcaaggctggcttggagcag gcccaggggccccccttctctactatgtaaaccaggcccgccaagctccccgactca agtatcttattggggctgcacctatagcctatggcttgcctgtctctctgcggtaaccc catggagctgtcttattgatgctagaagcctcataactgttctacctc 30 204933_s_at TNFRSF11B gataaaacggcaacacagctcacaagaacagactttccagctgctgaagttatgga aacatcaaaacaaagcccaagatatagtcaagaagatcatccaagatattgacctc tgtgaaaacagcgtgcagcggcacattggacatgctaacctcaccttcgagcagctt cgtagcttgatggaaagcttaccgggaaagaaagtgggagcagaagacattgaaa aaacaataaaggcatgcaaacccagtgaccagatcctgaagctgctcagtttgtggc gaataaaaaatggcgaccaagacaccttgaagggcctaatgcacgcactaaagca ctcaaagacgtaccactttcccaaaactgtcactcagagtctaaagaagaccatcag gttccttcacagc 31 204953_at SNAP91 agagaggtgctattcaagtgattctgaaggcaccccaaggtatatctgtaatttaaag attactgcaaatatctttactttactgtgggtttttagtacatctgttaatttagtgtttct ttgtgtgttttgtagactagtgttcttccatccttcaactgagctcaaagtaggttttgtt gtaacattgtgattaggatttaaactaattcagagaattgtatcttttactgtacatact gtattctttaagttttaatttgttgtcatactgtctgtgctgatggcttggcttaagatttt gatgcataaatgaggtcactgttgatcagtgttgctagtagcttggcagctcttcataa aagcatattgggttggaaaggtgtttgcctatttttca 32 205046_at CENPE aatcagcatctttccaatgaggtcaaaacttggaaggaaagaacccttaaaagaga ggctcacaaacaagtaacttgtgagaattctccaaagtctcctaaagtgactggaac agcttctaaaaagaaacaaattacaccctctcaatgcaaggaacggaatttacaag atcctgtgccaaaggaatcaccaaaatcttgtttttttgatagccgatcaaagtcttta ccatcacctcatccagttcgctattttgataactcaagtttaggcctttgtccagaggt gcaaaatgcaggagcagagagtgtggattctcagccaggtccttggcacgcctcctc aggcaaggatgtgcctgagtgcaaaactcagtagactcctctttgtcacttctctgga gatccagcattccttatttggaaatgactttgtttatgtgtctatccctggtaatgatgtt gtagtgcagcttaatttcaattcagtctttactttgccactag 33 205189_s_at FANCC ttccctccacctccaagacaggtggcggccgggcaggcactcttaagcccacctccc cctcttgttgccttcgatttcggcaaagcctgggcaggtgccaccgggaaggaatggc atcgagatgctgggcggggacgcggcgtggcgagggggcttgacggcgttggcggg gctgggcacaggggcagccgcagggaggcagggatggcaaggcgtgaagccacc ctggaaggaactggaccaaggtcttcagaggtgcgacagggtctggaatctgacctt actctagcaggagtttttgtagactctccctgatagtttagtttttgataaagcatgctg gtaaaaccactaccctcagagagagccaaaaatacagaagaggcggagagcgccc ctccaaccaggctgttattcccctggactc 34 205217_at TIMM8A gtacatgggactatgcttttctcaaagccccattaactgcttcctataattttgatagtg ggaccacatacgtaaaaatctctcatttgtgtggagtcatttctgatttcaggggagat ccttgtgtttatcagaaagggcagaagtaggggaagaataatttggtatccttatcta gtgtttgattgtcaatgctggagaaaaatatctgtaagagtgtttatacagtacacttc agttatcttgatctccctttcctatatgatgatttgcttaaatatccatattaagtaagtc tcaaggtagggtaggcagcctgagagtctagaggcctttagttataaaggaatctag ccagtgaacataattcttattactagactgccacaaggaagaaattaacttaccctgt atatcagggtacaaaaaattcagtgatgtgcctaaataagttataaagatttaggcc aatcagaagctaacagcagtttcaggtagaggtgcatgcctaatgttagttagtgta gattccatttactgcattctt 35 205386_s_at MDM2 tttcccctagttgacctgtctataagagaattatatatttctaactatataaccctagga atttagacaacctgaaatttattcacatatatcaaagtgagaaaatgcctcaattcac atagatttcttctctttagtataattgacctactttggtagtggaatagtgaatacttac tataatttgacttgaatatgtagctcatcctttacaccaactcctaattttaaataattt ctactctgtcttaaatgagaagtacttggttttttttttcttaaatatgtatatgacattt aaatgtaacttattattttttttgagaccgagtcttgctctgttacccaggctggagtgc agtgggtgatcttggctcactgcaagctctgccctccccgggttcgcaccattctcctg cctcagcctcccaattagcttggcctacagtcatctgcc 36 205433_at BCHE ggaaagcaggattccatcgctggaacaattacatgatggactggaaaaatcaattta acgattacactagcaagaaagaaagttgtgtgggtctctaattaatagatttaccctt tatagaacatattttcctttagatcaaggcaaaaatatcaggagcttttttacacacct actaaaaaagttattatgtagctgaaacaaaaatgccagaaggataatattgattcc tcacatctttaacttagtattttacctagcatttcaaaacccaaatggctagaacatgt ttaattaaatttcacaatataaagttctacagttaattatgtgcatattaaaacaatgg cctggttcaatttctttctttccttaataaatttaagttttttccccccaaaattatcagtg ctctgcttttagtcacgtgtattttcattaccactcgtaaaaaggtatcttttttaaatga attaaatattgaaacactgtacaccatagtttaca 37 205481_at ADORA1 gaggagaacactagacatgccaactcgggagcattctgcctgcctgggaacggggt ggacgagggagtgtctgtaaggactcagtgttgactgtaggcgcccctggggtgggt ttagcaggctgcagcaggcagaggaggagtacccccctgagagcatgtgggggaa ggccttgctgtcatgtgaatccctcaatacccctagtatctggctgggttttcaggggc tttggaagctctgttgcaggtgtccgggggtctaggactttagggatctgggatctgg ggaaggaccaacccatgccctgccaagcctggagcccctgtgttggggggcaaggt gggggagcctggagcccctgtgtgggagggcgaggcgggggagcctggagcccct gtgtgggagggcgaggcgggggatcctggagcccctgtgtcggggggcgagggag gggaggtggccgtcggttgaccttctgaacatgagtgtcaactccaggacttgcttcc aagcccttccctctgttggaaattgggtgtgccctggctcc 38 205491_s_at GJB3 tgcttccagccttcgtaattagacttcaccctgagtacacacacaatcactgccactct cactatagacaaaccacactccctcctctgtcacccagtcactgccatctcaacacac atccccaccctgtgtacacacaatctctgttattcatactctcactccttatgcgcactc tcaacagggcatgtagtctgcactcaagcatgccatcccagcctcaccctgcatttta ttcggctcatcccattttccctgaacattttcgctgaactagggccctggcaggatgct gggactgtgcaaggaggtaggacctatgcccacggagctaagagacaggaacaca ggctcatctcccgcactaaccaacccctgggatggctcacagcctgctcccagtgctg tgtcatgacctgaa 39 205501_at PDE10A atgcttgcccaacacactgtgaaatagttaccaaaatttgtacaaatgcagcatcttc attctttctgagaagacaagatggttttctttacatgaacaaatgaacaaaagagatc ctagatccataacgtagctaaggcatctaagagtttgctgttgataatcttgctgacc aaaaactactggagagtaacacaggttatatgccatcacaaatacaatgctcatga agaactgatttgtagagtcaatgaacctgtgtccagaattttaataggctctctattgg aaggagaaagaatttcaagttaacagtatctaactttatcatagttgatgttagtaaa ttttaaaaaatgattttatatgtatgacaaaaatctttgtaaaatgcgcaagtgcaat aatttaaagaggtcttaactttgcatttataaattataaatattgtacatgtgtgtaatt ttttcatgtattcatttgcagtctttgtatttaaaa 40 205825_at PCSK1 tttccattcccaatctagtgctagatgtataaatctttcttttgattcttcctaacaaaat attttctgggttaaaaccccagccaactcattgggttgtagccaaaggttcactctca agaagctttaatatttaaataaaatcatattgaatgtttccaacctggagtataatatt cagatataaaacagttttgtcagtctttcttagtgcctgtgtggatttttgtgaaaatgt caaagagaaaacttatatactatttcccttgaaattttaaactatattttctttacaggt atttataatataccaatgcttttatcaaacagaattttaaagagcataataaattatat taaagaaccaaaagttttcctgagaataagaaagtttcacccaataaaatatttttga aaggcatgttcctctgtcaatgaaaaaaagtacatgtatgtgttgtgatattaaaagt gacatttgtctaatagcctaatacaacatgtagctgagtttaacatgtgtggtcttg 41 205893_at NLGN1 gaacctaggagagtcaacatctggaggattttagtctttcttacacatatgtgtgattt taaacgaatattctcagaccacaggaaactcttcatccccctgttgtttaccagtaac agtatatcacagacctttccaaatgtttgtatatgtaatcagatgtacatttatattga aaaacaaatgagatggacttaaagagcacatcctgataaatactttctctctcacctg tactatatttctattagactaaagttatgtgattttttttttacattttttcagatgactag caattttgatagtttataagataatgcaaagaactttctctgacaaactaactgcagt aacagaaacctttcttttcagttactctttttcaagaatgaaagattattatacaaaaa attgtatactacttgatggaaccaactttgtacatcttggccatgtcactggtcattg 42 205938_at PPM1E catgctaggctttctcagtggggaaaaaaatggctggatagaactgggacaaacac agacccatctttaggggtctggattttgtaggtccgactacacagcagtgttaactcat ttctcatgccattagctctctacaaaataaagcaaagtagttctagtgtggtcgttata aaccaatattgtgaaaaatagcaactattcatttgttcacaacatgcgtatttataga gtagttaggtaccatttgtaaggtaaatcctttaaaattctataatacatactaaaata gtggttattggtctgatatatgctgctcttggttctataaactagataaaagcagtgct ttgtgaaatgcagtgttctctcttaacgccactggtgataggaagtagttcccttcagt tcaaatc 43 205946_at VIPR2 ttcctcccctgtagggtttggacagacccacccccagccttgcccagctttcaaagga caaaagggagcatcccccacctactctcaggtttttgaggaaacaaagatttgtggt aactgaaggtgttgggtcagtggccaggtgccgacactgagctgtgacccagaggg gacgctgaggaagtgggcgtgagtggacntgtcaggtggttaccaggcactggttgt tgatggtcggtggttgggtgtgggcagtcatcagtcatcaggtgtgctcaggggaca atctcccctcaaccgcacatgtgccactgttcagcggagctgactggtttcncctggt agagggnccggctgtttcctgacagatgcctggtgagcaggggaagcaggacccag tggtcancaggtgtctttaactgtcattgtgtgtggaatgtcgcagactcctccacgtg gcgggaatgagct 44 206043_s_at ATP2C2 gcaccacgacgatgacgttcacttgttttgtgtttttcgatctcttcaacgccttgacct gccgctctcagaccaagctgatatttgagatcggctttctcaggaaccacatgttcctc tactccgtcctggggtccatcctggggcagctggcggtcatttacatccccccgctgc agagggtcttccagacggagaacctgggagcgcttgatttgctgtttttaactggatt ggcctcatccgtcttcattttgtcagagctcctcaaactatgtgaaaaatactgttgca gccccaagagagtccagatgcaccctgaagatgtgtagtggaccgcactccgcggc accttccctaatcatctcgatctggttgtgactgtggcccctgccgtgtctcctcgtcag gggagacttttaggaggccgcagccttccatcaccggatcagtttttcctcttaggaa agctgcaggaacctcgtgggc 45 206096_at ZNF35 gtggctttcctaggaatgggtcgtacaaagctaagtggtaatgatgctatttggggaa aggtcttttttgcttaantttgttttttaaaactctgatgattncttgagcaacaggcag gttatctgcctggttgaattctggttgaaccgtgtattctaatatttctggttaagtggt gactgggtaaggaaaccacttggggtagcagttcaacaattcacttacgaatgtttat aagctttccatttcctaggtaattttttaaaagccagtcaaaacaaaaactttactgaa aatggacagaaataggaaatggactttttccttactgtctatacctcctgaaccttggt attgtaaagatctggggacctctgggtctgttctgaccattccctagtctccatggcca agcactcaaggattgatggacaccacacaccagctatattcatttgccaagatcaac agctccttctccaaacaactcaagcccccaattccnatcgcattcnnttngggtgag atgcaactaacagcccctt 46 206228_at PAX2 gcaggctagatccgaggtggcagctccagcccccgggctcgccccctngcgggcgt gccccgcgcgccccgggcggccgaaggccgggccgccccgtcccgccccgtagttg ctctttcggtagtggcgatgcgccctgcatgtctcctcacccgtggatcgtgacgactc gaaataacagaaacaaagtcaataaagtgaaaataaataaaaatccttgaacaaa tccgaaaaggcttggagtcctcgcccagatctctctcccctgcgagccctttttatttg agaaggaaaaagagaaaagagaatcgtttaagggaacccggcgcccagccaggc tccagtggcccgaacggggcggcgagggcggcgagggcgccgaggtccggcccat cccagtcctgtggggctggccgggcagagaccccggacccaggcccaggcctaacc tgctaaatgtccccggacggttctggtctcctcggccactttcagtgcgtcggttcgttt tgattctttt 47 206232_s_at B4GALT6 tgcagttttgcatgtaatcggttatacctttattggacttttatagacattttttatttgca tgaaaaaaactcactaaatttacatcactaaacaaaggttaacccttgtgtgaaatg aaggaactgtcaataattgacagccaactaatacagtaaactgttatactagttttga gctttagacctcagccttttgtgtggaagaagtcacagctttcttaggctttaaaggaa aagaaggaaggacttaaatagcttttcttcctaccgggattacctatgtttttccttgct tgcaatctcatctgattttgctagaaatcacaaccatattgtttatgcatattgcatga gtattaccaagaaaaaaatctttaaaagttgtgatgtgacatgatataaaggatctct ttatgttaaatgtctttccatgtacctctggtgtgtcagggattttgtgcctcaaaaaat gtttccaaggttgtgtgtttatactgtgtattttttttaaattcacggtgaacagcacttt tattatttcca 48 206401_s_at MAPT aggtggcagtggtccgtactccacccaagtcgccgtcttccgccaagagccgcctgc agacagcccccgtgcccatgccagacctgaagaatgtcaagtccaagatcggctcc actgagaacctgaagcaccagccgggaggcgggaaggtgcaaatagtctacaaac cagttgacctgagcaaggtgacctccaagtgtggctcattaggcaacatccatcata aaccaggaggtggccaggtggaagtaaaatctgagaagcttgacttcaaggacag agtccagtcgaagattgggtccctggacaatatcacccacgtccctggcggaggaaa taaaaagattgaaacccacaagctgaccttccgcgagaacgccaaagccaagaca gaccacggggcggagatcgtgtacaagtcgccagtggtgtctggggacacgtctcca cggcatctcagcaatgtctcctccaccggcagcatcgacatggtagactcgccccag ctcgccacgctagctgacgaggtgtctgcctcc 49 206426_at MLANA gtaaagatcctatagctctttttttttgagatggagtttcgcttttgttgcccaggctgg agtgcaatggcgcgatcttggctcaccataacctccgcctcccaggttcaagcaattc tcctgccttagcctcctgagtagctgggattacaggcgtgcgccactatgcctgacta attttgtagttttagtagagacggggtttctccatgttggtcaggctggtctcaaactcc tgacctcaggtgatctgcccgcctcagcctcccaaagtgctggaattacaggcgtga gccaccacgcctggctggatcctatatcttaggtaagacatataacgcagtctaatta catttcacttcaaggctcaatgctattctaactaatgacaagtattttctactaaacca gaaattggtagaaggatttaaataagtaaaagctactatgtactgccttagtgctgat gcctgtgtactgccttaaatgtacctatggcaatttagctctcttgggttcccaaatccc tctcacaagaatgt 50 206496_at FMO3 aaagcccaacatcccatggctgtttctcacagatcccaaattggccatggaagtttat tttggcccttgtagtccctaccagtttaggctggtgggcccagggcagtggccaggag ccagaaatgccatgctgacccagtgggaccggtcgttgaaacccatgcagacacga gtggtcgggagacttcagaagccttgcttctttttccattggctgaagctctttgcaatt cctattctgttaatcgctgttttccttgtgttgacctaatcatcattttctctaggatttct gaaagttactgacaatacccagacaggggctttgc 51 206505_at UGT2B4 taattacgtctgaggctggaagctgggaaacccaataaatgaactcctttagtttatt acaacaagaagacgttgtgatacaagagattcctttcttcttgtgacaaaacatcttt caaaacttaccttgtcaagtcaaaatttgttttagtacctgtttaaccattagaaatatt tcatgtcaaggaggaaaacattagggaaaacaaaaatgatataaagccatatgag gttatattgaaatgtattgagcttatattgaaatttattgttccaattcacaggttacat gaaaaaaaatttactaagcttaactacatgtcacacattgtacatggaaacaagaac attaagaagtccgactgacagtatcagtactgttttgcaaatactcagcatactttgg atccatttcatgcaggattgtgttgttttaac 52 206524_at T agcagtggaggagcacacggacctttccccagagcccccagcatcccttgctcacac ctgcagtagcggtgctgtccaggtggcttacagatgaacccaactgtggagatgatg cagttggcccaacctcactgacggtgaaaaaatgtttgccagggtccagaaacttttt ttggtttatttctcatacagtgtattggcaactttggcacaccagaatttgtaaactcca ccagtcctactttagtgagataaaaagcacactcttaatcttcttccttgttgctttcaa gtagttagagttgagctgttaaggacagaataaaatcatagttgaggacagcaggtt ttagttgaattgaaaatttgactgctctgccccctagaatgtgtgtattttaagcatatg tagctaatctcttgtgtt 53 206552_s_at TAC1 ttcagcttcatttgtgtcaatgggcaatgacaggtaaattaagacatgcactatgagg aataattatttatttaataacaattgtttggggttgaaaattcaaaaagtgtttattttt catattgtgccaatatgtattgtaaacatgtgttttaattccaatatgatgactccctta aaatagaaataagtggttatttctcaacaaagcacagtgttaaatgaaattgtaaaa cctgtcaatgatacagtccctaaagaaaaaaaatcattgctttgaagcagttgtgtca gctactgcggaaaaggaaggaaactcctgacagtcttgtgcttttcctatttgttttca tggtgaaaatgtactgagattttggtattacactgtatttgtatctctgaagcatgtttc atgttttgtgactatatagagatgtttttaaaagtttcaatgtgattctaatgtcttcatt tcattgtatgatg 54 206619_at DKK4 ctgtctgacacggactgcaataccagaaagttctgcctccagccccgcgatgagaag ccgttctgtgctacatgtcgtgggttgcggaggaggtgccagcgagatgccatgtgct gccctgggacactctgtgtgaacgatgtttgtactacgatggaagatgcaaccccaat attagaaaggcagcttgatgagcaagatggcacacatgcagaaggaacaactggg cacccagtccaggaaaaccaacccaaaaggaagccaagtattaagaaatcacaag gcaggaagggacaagagggagaaagttgtctgagaacttttgactgtggccctgga ctttgctgtgctcgtcatttttggacgaaaatttgtaagccagtccttttggagggaca ggtctgctccagaagagggcataaagacactgctcaagctccagaaatcttccagcg ttgcgactgtggccctggactactgtgtcgaagccaattgaccagcaatcggcagca tgctcgat 55 206622_at TRH gccctcttcctttaggcatgtgagaaaatcagcctagcagtttaaaccccactttcctc cacttagcaccataggcaagggggcagatcccagagcccctctcaccccccccacc acaggcctgctccttccttagccttggctaagatggtccttctgtgtcttgcaaagact ccccaagtggacagggagcccctgggagggcagccagtgagggtggggtgggact gaagcgttgtgtgcaaatccagcttccatcccctccccaacctggcaggattctccat gtgtaaacttcacccccaggacccaggatcttctcctttctgggcatccctttgtgggt gggcagagccctgacccacagctgtgttactgcttggagaagcatatgtaggggcat accctgtggtgttgtgctgtgtctggctgtgggataaatgtgtgtgggaatattgaaac atcgcctaggaattgtggtttgtatataaccctctaagcccctatcccttgtcgatgac agtca 56 206661_at DBF4B accaggagtgtcagcttttagaaggatcatggtcatgtgagcttctggtcaccggaag ccagaaatactcagctgccatgttgatccacaaaggtgggaggatgtggggaaggg ggaaagcggtgaggacgcagagtgcaggctgtggcctcggcatcccgcaggaggtc cctagaacatgccgtttcatgtcacctgctacagctctcccccagctagtatgatgatc cgttttacaaatgcagaaatgatcttaatattcatgaccactggccaggcgaggtggc tcacacctgtaatcccagcactttgggaggccaaggcgggtggatcacaaggtcaa gagttcgagaccagcctgaccaacgtggtgaaaccccgtctctactaaaaatagaa gcattagccgagcctggtgg 57 206672_at AQP2 gcgcagagtagctgcttcctggacgtgcgcgcccaggccagtgctgtgagcaggcg gggaggaggctgccggaggagcctgagcctggcaggttcccctgccctgaggctgt gagcagctagtggtggcttctcctgcctttttcagggaactgggaaacttaggggact gagctggggagggaggcaggtgggtggtaagagggaaactctggagagcctgcac ccaggtactgagtggggagtgtacagaccctgccttgggggttctgggaatgatgca actggttttactagtgtgcaagtgtgttcatccccaagttctcttttgtcctcacatgca gagttgtgcatgcccctgagtgtgaacaggtttgcctacgttggtgca 58 206678_at GABRA1 tggtttattgccgtgtgctatgcctttgtgttctcagctctgattgagtttgccacagtaa actatttcactaagagaggttatgcatgggatggcaaaagtgtggttccagaaaagc caaagaaagtaaaggatcctcttattaagaaaaacaacacttacgctccaacagca accagctacacccctaatttggccaggggcgacccgggcttagccaccattgctaaa agtgcaaccatagaacctaaagaggtcaagcccgaaacaaaaccaccagaaccca agaaaacctttaacagtgtcagcaaaattgaccgactgtcaagaatagccttcccgc tgctatttggaatctttaacttagtctactgggctacgtatttaaacagagagcctcag ctaaaagcccccacaccacatcaatagatcttttactcacattctgttgttcagttcctc tgcactgggaatttatttatgttctcaacgcagtaattccca 59 206799_at SCGB1D2 tagaagtccaaatcactcattgtttgtgaaagctgagctcacagcaaaacaagccac catgaagctgtcggtgtgtctcctgctggtcacgctggccctctgctgctaccaggcca atgccgagttctgcccagctcttgtttctgagctgttagacttcttcttcattagtgaac ctctgttcaagttaagtcttgccaaatttgatgcccctccggaagctgttgcagccaag ttaggagtgaagagatgcacggatcagatgtcccttcagaaacgaagcctcattgcg gaagtcctggtgaaaatattgaagaaatgtagtgtgtgacatgtaaaaactttcatcc tggtttccactgtctttcaatgacaccctgatctt 60 206835_at STATH aagcttcacttcaacttcactacttctgtagtctcatcttgagtaaaagagaacccagc caactatgaagttccttgtctttgccttcatcttggctctcatggtttccatgattggagc tgattcatctgaagagaaatttttgcgtagaattggaagattcggttatgggtatggc ccttatcagccagttccagaacaaccactatacccacaaccataccaaccacaatac caacaatataccttttaatatcatcagtaactgcaggacatgattattgaggcttgatt ggcaaatacgacttctacatccatattctcatctttcataccatatcacactactacca ctttttgaagaatcatcaaagagcaatgcaaatgaaaaacactataatttactgtata ctctttgtttcaggatacttgccttttcaattgtcacttgatgatataattgcaatttaaa ctgttaagctgtgttcagtactgtttc 61 206940_s_at LOC100131317 ggtttgttaccatcctttaatcataactaaaacattgaaaacagaacaaatgagaaa /// agaaaaaaaacctgccgattaacaatgacgaaaatcatgcatgatctgaaaggtgt POU4F1 ggaaagaaacacaattaggtctcactctggttaggcattatttatttaattatgttgta tatcattgtttgcagggcaacattctatgcattgaactgagcactaactgggctagctt ctggtagacgtttgtggctagtgcgattcacagtctactgcctgttccactgaaacatt ttgtcatattcttgtattcaaagaaaaaaggaaaaaaagattattgtaaatattttatt taatgcacacattcacacagtggtaacagactgccagtgttcatcctgaaatgtctca cggattgatctacctgtccatgtatgtctgctgagctttctccttggttatgttttt 62 206984_s_at RIT2 taaagagctcatttttcaggtccgccacacctatgaaattcccctggtgctggtgggta acaaaattgatctggaacagttccgccaggtttctacagaagaaggcttgagtcttgc ccaagaatataattgtggtttttttgagacctctgcagccctcagattctgtattgatga tgcttttcatggcttagtgagggaaattcgcaagaaggagtccatgccatccttgatg gaaaagaaactgaagagaaaagacagcctgtggaagaagctcaaaggttctttga agaagaagagagaaaatatgacatgatatctttgcttttgagttcctcacgctctctg aattttattagttggacaattccatatgtagcattctgcttcaatattatctctctatgtg tctctctctctttaaatatctgcctgtaggtaaaagcaagctctgcatatctgtacctct tgagatagttttgttttgcctttaacagttggatgga 63 207003_at GUCA2A gaggggtcaccgtgcaggatggaaatttctccttttctctggagtcagtgaagaagct caaagacctccaggagccccaggagcccagggttgggaaactcaggaactttgcac ccatccctggtgaacctgtggttcccatcctctgtagcaacccgaactttccagaaga actcaagcctctctgcaaggagcccaatgcccaggagatacttcagaggctggagg aaatcgctgaggacccgggcacatgtgaaatctgtgcctacgctgcctgtaccggat gctaggggggcttgcccactgcctgcctcccctccgcagcagggaagctcttttctcct gcagaaagggccacccatgatactccactcccagcagctcaacctaccctggtccag tcgggaggagcagcccggggaggaactgggtgact 64 207028_at LOC100129296 ctccccccgagagaaggctgcaaagctgggaagcccagggtgtgctcctcccgccct /// tttggacccccgggcttgcaccggctgcactctgagaaccagctgcgcgcggagcgg MYCNOS tgcaatgcagcacccaccctgcgagcctggcaattgcttgtcattaaaagaaaaaaa aattacggagggctccgggggtgtgtgttggggaggggagaccgatgcttctaaccc agcccccgctttgactgcgtgttgtgcagctgagcgcgaggccaacgttgagcaagg ccttgcagggaggttgctcctgtgtaattacgaaagaaggctagtccgaaggtgcaa aatagcagggagaggacgcgcccccttaggaacaagacctctggatgtttccagttt caaattgaaagaagaggggcgccccccttg 65 207208_at RBMXL2 acagcagcagttatggccggagcgaccgctactcgaggggccgacaccgggtgggc agaccagatcgtgggctctctctgtccatggaaaggggctgccctccccagcgtgatt cttacagccggtcaggctgcagggtgcccaggggcggaggccgtctaggaggccgc ttggagagaggaggaggccggagcagatactaagcaggaacagacttgggaccaa aaatcccttttcaacgaaactaacaaaaagaagaacctgttgtatggtaactaccca aggactagtacaaggaagagttgtttttaccttttaagaatttcctgttaagatcgtct ccatttttatgcttttgggagaaaaaacttaaaattcgtttagtttagttttggaattgtt aacgtttctttcaacaagctcctgttaaaagtatatgaacctgagtactagtcttctta catttacaagtagaaattcgattaatggcttcttcccttgtaaattttcttg 66 207219_at ZNF643 cagccagagcattggactgatccagcatttgagaactcatgttagagagaaacctttt acatgcaaagactgtggaaaagcgtttttccagattagacaccttaggcaacatgag attattcatactggtgtgaaaccctatatttgtaatgtatgtagtaaaaccttcagcca tagtacatacctaactcaacaccagagaactcatactggagaaagaccatataaat gtaaggaatgtgggaaagcctttagccagagaatacatctttctatccatcagagag tccatactggagtaaaaccttatgaatgcagtcattgtgggaaagcctttaggcatga ttcatcctttgctaaacatcagagaattcatactggagaaaaaccttatgattgtaat gagtgtggaaaagccttcagctgtagttcatcccttattagacactgcaaaacacatt taagaaataccttcagcaatgttgtgtgaaatatactaaacatcaaagaatctatgtt ggagcacaagattctaaatcagtggttccctg 67 207529_at DEFA5 gagtcactccaggaaagagctgatgaggctacaacccagaagcagtctggggaag acaaccaggaccttgctatctcctttgcaggaaatggactctctgctcttagaacctc aggttctcaggcaagagccacctgctattgccgaaccggccgttgtgctacccgtga gtccctctccggggtgtgtgaaatcagtggccgcctctacagactctgctgtcgctga gcttcctagatagaaaccaaagcagtgcaagattcagttcaaggtcctgaaaaaag aaaaacattttactctgtgtaccttgtgtctt 68 207597_at ADAM18 gtgacgctcaatctacagtttattcatatattcaagaccatgtatgtgtatctatagcc actggttcctccatgagatcagatggaacagacaatgcctatgtggctgatggcacc atgtgtggtccagaaatgtactgtgtaaataaaacctgcagaaaagttcatttaatgg gatataactgtaatgccaccacaaaatgcaaagggaaagggatatgtaataattttg gtaattgtcaatgcttccctggacatagacctccagattgtaaattccagtttggttcc ccagggggtagtattgatgatggaaattttcagaaatctggtgacttttatactgaaa aaggctacaatacacactggaacaactggtttattctgagtttctgcatttttctgccg tttttcatagttttcaccactgtgatctttaaaagaaatgaaataagtaaatcatgtaa cagagagaatgcagagtataatcgtaattcatccgttgtatcag 69 207814_at DEFA6 gagccactccaagctgaggatgatccactgcaggcaaaagcttatgaggctgatgc ccaggagcagcgtggggcaaatgaccaggactttgccgtctcctttgcagaggatgc aagctcaagtcttagagctttgggctcaacaagggctttcacttgccattgcagaagg tcctgttattcaacagaatattcctatgggacctgcactgtcatgggtattaaccacag attctgctgcctctgagggatgagaacagagagaaatatattcataatttactttatg acctagaaggaaactgtcgtgtgtcccatacattgccatcaactttgtttcctcat 70 207843_x_at CYB5A gctggaggtgacgctactgagaactttgaggatgtcgggcactctacagatgccagg gaaatgtccaaaacattcatcattggggagctccatccagatgacagaccaaagtta aacaagcctccagaaccttaaaggcggtgtttcaaggaaactcttatcactactattg attctagttccagttggtggaccaactgggtgatccctgccatctctgcagtggccgtc gccttgatgtatcgcctatacatggcagaggactgaacacctcctcagaagtcagcg caggaagagcctgctttggacacgggagaaaagaagccattgctaactacttcaac tgacagaaaccttcacttgaaaacaatgattttaatatatctctttctttttcttccgac attagaaacaaaacaaaaagaactgtcctttctgcgctcaaatttttcgagtgtgcct ttttattcatctacttt 71 207878_at KRT76 gagctcaagccagcatagctccaccaagtgatctactgttccaaatctctataaccac ctgcttcccactcagcctgcaatagtgtttcccactctctgcttggcatcaatagatgc ataagggtcaaccacatttttcctcaagttccctggagaagaagctgaactcctggtt tctccatccccatgaccttcccagggccatggaggtcctgctgctggtctgggatgat gatgcccctggaaaccttcctgcaatggccccttactttggacagcaacccctgagcc caagccagttttggccttcacagcctggccggttcccactctggcccatctcccattctt actgggagttggagatttgaagccagtcatctcagcactgtctgaggagggcagagc catgggttctgtgctggagggtgcacggccaagatctccagactgctggttcccagg gaaccctccctacatctgggcttcagatcctgactcccttctgtcccctaattccctga gctgtagatcctctggt 72 207937_x_at FGFR1 cgcacccgcatcacaggggaggaggtggaggtgcaggactccgtgcccgcagactc cggcctctatgcttgcgtaaccagcagcccctcgggcagtgacaccacctacttctcc gtcaatgtttcagcttgcccagatctccaggaggctaagtggtgctcggccagcttcc actccatcactcccttgccatttggacttggtactcggcttagtgattagaggccctga acaggtggtggtatccctgctctgctggagaggaacccagatgctctcccctcctcgg aggatgatgatgatgatgatgactcctcttcagaggagaaagaaacagataacacc aaaccaaaccccgtagctccatattggacatccccagaaaagatggaaaagaaatt gcatgcagtgccggctgccaagacagtgaagttcaaatgcccttccagtgggacccc aaaccccacactgcgctggttgaaaaatggcaaagaattcaaacctgaccacagaa ttggaggctacaaggtccgttatgccacctgga 73 208157_at SIM2 ctgccctgtacatgctagttcaacagaaaggaatggcctttcaccttctcctggtggc aggcaagcagatgtcctctgcggagataccgccagctccccaggacgcagactgac tcctgtttgctcgctggaccaaccccaggcagaaggtggaaggtgggaacagaggtt tagctgcaggacatgtattcccattgcaccgagacctaactgccgctcagagtgtag accgagatggtgcagatgcctgcagtgccattaaaatgtgggtgaaggtgacatcag gattatgtgccccaggccgggctcagtggctcacacctgtaatcccagcactttggga ggccaaggtgggcggatcacctgaggtcaggagtttgcgacaagcctgccaacaag ctgaaacc 74 208233_at PDPN gaaatctctgatataagctgggtgtggtggctcgtgcctgtagtctcagctgctgggc aactgcagaccagcctgggcaacatagtaagaccctgtctcaaaaaaataatctctg gtacaatggtcatgttccaaagttccttacttgggcctcttgagtgcagtggctcacac ctggaatcccagtgctttgagaggctgaggaggcaggaggttcacttgtgcccagga atttgaggctgcagtgagctatgattgtgccactgcactccagcctgggtgacagagc aagactgtgctctcttaaaaataagaaagagcctcttcatcttcaaaaggactacatc tgaagtttccccagaaggacaaatgtctacttagaccttataaatttccaaaataaga gagtcagagccagaggtggcttgtaagttgacttctgttgagatctgaccacatttga tctcttgttttaattttccaactaactgaacttggaagaaaacccaaaccaagttttaa tctgatgccta 75 208292_at BMP10 ccatgagcaacttccagagctggacaacttgggcctggatagcttttccagtggacct ggggaagaggctttgttgcagatgagatcaaacatcatctatgactccactgcccga atcagaaggaacgccaaaggaaactactgtaagaggaccccgctctacatcgactt caaggagattgggtgggactcctggatcatcgctccgcctggatacgaagcctatga atgccgtggtgtttgtaactaccccctggcagagcatctcacacccacaaagcatgc aattatccaggccttggtccacctcaagaattcccagaaagcttccaaagcctgctgt gtgcccacaaagctagagcccatctccatcctctatttagacaaaggcgtcgtcacct acaagtttaaatacgaaggcatggccgtctccgaatgtggctgtagatagaagaag agtcctatggcttatttaataactgtaaatgtgtatatttggtgttcctatttaatgaga ttatttaataagggtgtacagtaatagaggcttgctgccttcaggaa 76 208314_at RRH atgatctgcatgtttctggtggcatggtccccttattccatcgtgtgcttatgggcttctt ttggtgacccaaagaagattcctccccccatggccatcatagctccactgtttgcaaa atcttctacattctataacccctgcatttatgtggttgctaataaaaagtttcggaggg caatgcttgccatgttcaaatgtcagactcaccaaacaatgcctgtgacaagtatttt acccatggatgtatctcaaaacccattggcttctggaagaatctgaaataagagaaa aggacacgctatcaaaacactttagttttttgacaatgcttttcttttaaatatgagccc atttagatcaagtgcagacatggatcattgtcctatgagagtgtaagctcctcaagca cagctcgtgcttccgtttgtgcactctggctgctgtagtgtatgcttctctgtgtcctgat atatcaacttattgctcatctcctttgatgaattaggcatcagaggttaaggtcccctttc 77 208368_s_at BRCA2 gaacaggagagttcccaggccagtacggaagaatgtgagaaaaataagcaggaca caattacaactaaaaaatatatctaagcatttgcaaaggcgacaataaattattgac gcttaacctttccagtttataagactggaatataatttcaaaccacacattagtactta tgttgcacaatgagaaaagaaattagtttcaaatttacctcagcgtttgtgtatcggg caaaaatcgttttgcccgattccgtattggtatacttttgcttcagttgcatatcttaaa actaaatgtaatttattaactaatcaagaaaaacatctttggctgagctcggtggctc atgcctgtaatcccaacactttgagaagctgaggtgggaggagtgcttgaggccagg agttcaagaccagcctgggcaacatagggagacccccatctttacgaagaaaaaaa aaaaggggaaaagaaaatcttttaaatctttggatttgatcactacaagt 78 208399_s_at EDN3 ccgagccgagcttactgtgagtgtggagatgttatcccaccatgtaaagtcgcctgcg caggggagggctgcccatctccccaacccagtcacagagagataggaaacggcatt tgagtgggtgtccagggccccgtagagagacatttaagatggtgtatgacagagcat tggccttgaccaaatgttaaatcctctgtgtgtatttcataagttattacaggtataaa agtgatgacctatcatgaggaaatgaaagtggctgatttgctggtaggattttgtaca gtttagagaagcgattatttattgtgaaactgttctccactccaactcctttatgtggat ctgttcaaagtagtcactgtatatacgtatagagaggtagataggtaggtagatttta aattgcattctgaatacaaactcatactccttagagcttgaattacatttttaaaatgc atatgtgctgtttggcaccgtggcaagatggtatcagagagaaacccatcaattgctc aaatactc 79 208511_at PTTG3 ttgtggctacaaaggatgggctgaagctggggtctggaccttcaatcaaagccttag atgggagatctcaagtttcaatatcatgttttggcaaaacattcgatgctcccacatcc ttacctaaagctaccagaaaggctttgggaactgtcaacagagctacagaaaagtc agtaaagaccaatggacccctcaaacaaaaacagccaagcttttctgccaaaaaga tgactgagaagactgttaaagcaaaaaactctgttcctgcctcagatgatggctatcc agaaatagaaaaattatttcccttcaatcctctaggcttcgagagttttgacctgcctg aagagcaccagattgcacatctccccttgagtgaagtgcctctcatgatacttgatga ggagagagagcttgaaaagctgtttcagctgggccccccttcacctttgaagatgcc ctctccaccatggaaatccaatctgttgcagtctcctttaagcattctgttgaccctgg atg 80 208684_at COPA ggtttaaggatcagtcctctgcagtttcgctaaggccccctttgtgtgcatgggtcagt caccatatgttccccccagagaatgtgtctatatcctccttctaacagcaccttccccc tgcagctactcttcagatctggctctctgtaccctaaaacctagtatctttttctcttcta tggaaaatccgaaggtctaaacttgacttttttgaggtcttctcaacttgactacagtt gtgctcataattgtccttgcctttccagcttaattattttaaggaacaaatgaaaactct gggctgggtggagtggctcatacctgtaatcccagcactttgggaggctacggtggg cagatcatctgaggccaggagttcgagacctgcctggccaacatggcaacaccccgt ctctaataaaaatataaaaattagcctggcatggtagcatgcgcctatagtcccagct gctcaggaggctgaggcatgagaatcgcttgaacctaggaggtggaggttgcattca actgagatcatacc 81 208992_s_at STAT3 actggtctatctctatcctgacattcccaaggaggaggcattcggaaagtattgtcgg ccagagagccaggagcatcctgaagctgacccaggcgctgccccatacctgaagac caagtttatctgtgtgacaccaacgacctgcagcaataccattgacctgccgatgtcc ccccgcactttagattcattgatgcagtttggaaataatggtgaaggtgctgaaccct cagcaggagggcagtttgagtccctcacctttgacatggagttgacctcggagtgcg ctacctcccccatgtgaggagctgagaacggaagctgcagaaagatacgactgagg cgcctacctgcattctgccacccctcacacagccaaaccccagatcatctgaaactac taactttgtggttccagattttttttaatctcctacttctgctatctttgagc 82 209434_s_at PPAT ttgacagctctttaagcccacatgcagcagtgggtcagataaccctgtggcagtgac acgggcaaattggcatttgaataaagccctgggaccacctcaacatgcgtagcctct tgtcttaaatgtactccccatggcagcatggaggaggcaagacctgtgggtcaatttt gaactggccttactttgatttttaaaacaagagactcagggaaagtactaaaccaaa atctctgattttactttgcgttttctgtagtttttgttttactgagatgcttttgtaaagga aaataatactgtgacagtttagtaattctacagattcttaatatttctccatcatggcct tttacttcacaattttctgaagtctgaattcaattacaattttttttttttaccaatttaat ctcaaatgttgtttaactgctttaaattcatatacgtagagtattataaactgcagaga tgaaaaatgtgttttcacgggatttatattgtgaactaaactaagcctactttttgtga ct 83 209839_at DNM3 gagacttctcacttctggttggaggtttcacatatggctcaactcaagtcattaatctct ttttaatttttactcttgaattccttaaacttcgctcattatgaaatgttttaaaattatg acaaaaattactctgtctaaccacttgccttgtctgctaccagtttgttaaaaattattc cccccaaccagtaattccaccagtactacttgatttgtgttatatttcctatgtacatgt acagcctttgttttgcttgcttgtctatttttactttcccttttttgggtcaaatttttctttt gctttgtttgaagaaggaatatacagaagtaaaatcttgtcttctctgctgattcttta attaatatgagccggatactttccactgtcttcttggcactttcaggatttcttaatgct gatatatggactcttagaatggaatttttgaagaaaaatctcaaagcctgtatcgttct 84 209859_at TRIM9 ataggttacccttgaaattcattagtttgtcataaagttttaggaaaggtaggacccg gaaagaagttctaattagttgtctaaatatttttcagtgagccaagaaattcaccatg aaaaaacaagaataacaaatagaagggaagagataggatgggaaagctaacaaa ttaaagttttggcaaaaaggaatatatgtaaatagctaattatttacttttgtgcttact ttatttagattatttctatcagttacaatctttttctagttaagtgtacctaatttatgga atgggtgctatcctgtttatgtgtgtcttggtttttcttggctacagaaaaactgttgca gggcaacactagtttgatatttgatttactctccaatgagactcaatggctgggccgt ggtagactcatagttcctcttgttctttattaaattcatcctgctaattagatttctagtg acttgtaacatgtagtttacactgaattgcaattacagatgcatacaactactatacta 85 210016_at LOC100134306 ataacagcatatgcatttccccaccgcgttgtgtctgcagcttctttgccaatatagta /// atgcttttagtagagtactagatagtatcagttttggattcttattgttatcacctatgta MYT1L caatggaaagggattttaagcacaaacctgctgctcatctaacgttggtacataatct caaatcaaaagttatctgtgactattatatagggatcacaaaagtgtcacatattaga atgctgacctttcatatggattattgtgagtcatcagagtttattataacttattgttcat attcatttctaagttaatttaagtaatcatttattaagacagaattttgtataaactatt tattgtgctctctgtggaactgaagtttgatttatttttgtactacacggcatgggtttgt tgacactttaattttgctataaatgtgtggaatcacaagttgctgtgatacttcattttt aaattgtgaactttgtacaaattttgtcatgctggatgttaacacat 86 210247_at SYN2 tcatgtcttattcttccctgtgaaaccaggattaatcgtggactcctggcagcttaacc tagctcagttgcagtgctaagcatgccccgcccccattcagtgatacctgtttgggaa gtatatacttccccaaaagtactcttggccctaagttttaggaactttccccgacctgg atcccttgtcatacctgtgttactgtttaaagcacacccacccaacttacaagatctta ggctgctgtggtggtgaagcaccttgagtctgctgatattcgggagaacaaggatct gcagtttccccttttctcccctctgaagagtggttcttatgtgcaatctgcagtaacctt gaactccagagctgcactatagaggagaatgcatgccactatgacagcagtatgcc aagctttgtgttcatctcctaata 87 210302_s_at MAB21L2 atttcgttttgcttttggttgcctgaatgttgtcaccaagtgaaaaaattatttaactat atgtaaaatttctcttttaaaaaaaagttttactgatgttaaacgttctcagtgccaat gtcagactgtgctcctccctctcctgaacctctaccctcaccctgagctgtcttgttgaa aacagt 88 210315_at SYN2 tattctcgactgtaatggcattgcagtagggccaaaacaagtccaagcttcttaaaat gattggtggttaatttttcaaagcagaaattttaagccaaaaacaaacgaaaggaaa gcggggaggggaaaacagaccctcccactggtgccgttgctgcgttctttcaatgctg actggactgtgtttttcctatgcagtgtcagctcctctgtctggttgtttacctgttcctgt tcgtgcttgtaatgctcacttatgttttctctgtataacttgtgattccagggctgtttgt caacagtatacaaaagaattgtgcctctcccaagtccagtgtgactttatcttctgggt ggtttg 89 210455_at C10orf28 gaaatcagcgaggctcaagttccaagcaaaccattccaaaatgtggaattctgtgac ttcagtaggcatgaacctgatggggaagcatttgaagacaaagatttggaaggcag aattgaaactgataccaaggttttggagatactatatgagtttcctagagtttttagtt ctgtcatgaaacctgagaatatgattgtaccaataaaactaagctctgattctgaaat tgtacaacaaagcatgcaaacatcagatggaatattgaatcccagcagcggaggca tcaccactacttctgttcctggaagtccagatggtgtctttgatcaaacttgcgtagatt ttgaagttgagagtgtaggtggtatagccaatagtacaggtttcatcttagatcaaaa gatacagattccattcctgcaactatgggtcacatctctctgtcagagagcacaaatg acactgttagtccagtaatgattagagaatgtgagaagaatgacagcactgctgatg agttacatgtaaagcacgaacctcctgatacag 90 210758_at PSIP1 gggctcaaagcattaatccagttactgaaaagagaatacaagtggagcaaacaag agatgaagatcttgatacagactcattggactgaatttcccccttccccccatgatgg aagaatgttcagattctaaattgaggacttcattattaatggcattactgtgttatgatt aacaaatttcttgtaaggtacacactacatactaaggtcggccatcattccgtttttttt tttttttttttttttaaccaagcttaaaatgaagcttaaaatgaagctttgtgtttgaaag taataacaagctcagacgaagatggtggttgtacattattcatctagaaaatataaa aattcattttgttttgaagctagttattaaactggaatagcagttatatccctgagaat ggggccctt 91 210918_at — gctgctgttttcttctaactgcagggaaaatgctgtctaaaagaaaataataaatttgt atctgctgagttctcttagcataaggcaccaacaaaacaaccttcaggaagggaga agaaaccatcctcccactcatccttcagaggatttagataaagtgaagggaagaatc gttctccagctccttcggaatttacgccggcatcagggcaggcttgttactgctggatc cattgtctgctcaaggttacttattccactaagacgtacatcctaccacggaccacgg ctttgtagctagccaggctctgagtgtgtgtgtagatgaaccatttctctctccagtaa atgaatgacagtctttctagggctcttgtcttctgctgggaggcag 92 211204_at ME1 agtcactctcccagatggacggactctgtttcctggccaaggcaacaattcctacgtg ttccctggagttgctcttggggtggtggcctgcggactgagacacatcgatgataagg tcttcctcaccactgctgaggtcatatctcagcaagtgtcagataaacacctgcaaga aggccggctctatcctcctttgaataccattcgagacgtttcgttgaaaattgcagtaa agattgtgcaagatgcatacaaagaaaagatggccactgtttatcctgaaccccaaa acaaagaagaatttgtctcctcccagatgtacagcactaattatgaccagatcctacc tgattgttatccgtggcctgcagaagtccagaaaatacagaccaaagtcaaccagta acgcaacagcta 93 211264_at GAD2 gttccacttctctaggtagacaattaagttgtcacaaactgtgtgaatgtatttgtagtt tgttccaaagtaaatctatttctatattgtggtgtcaaagtagagtttaaaaattaaac aaaaaagacattgctccttttaaaagtcctttcttaagtttagaatacctctctaagaa ttcgtgacaaaaggctatgttctaatcaataaggaaaagcttaaaattgttataaata cttcccttacttttaatatagtgtgcaaagcaaactttattttcacttcagactagtagg actgaatagtgccaaattgcccctgaatcataaaaggttctttggggtgcagtaaaa aggacaaagtaaatataaaatatatgttgacaataaaaactcttgcctttttcatagt attagaaaaaaatttctaatttacctatagcaacatttcaaat 94 211341_at LOC100131317 gcatttgaaactgagcactaaactgggctagctttctggtagaccgttttgtggctagt /// gcgatttcacagtctactgcctgtttccactgaaaacatttttgtcatattcttgtattca POU4F1 aagaaaacaggaaaaaagttattgtaaatattttatttaatgcacacattcacacag tggtaacagactgccagtgttcatcctgaaatgtctcacggattgatctacctgtctat gtatgtctgctgagctttctccttggttatgttttttctcttttacctttctcctcccttactt ctatcagaaccaattctatgcgccaaatacaacagggggatgtgtcccagtacactt acaaaataaaacataactgaaagaagagcagttttatgatttgggtgcgtttttgtgt ttatactgggccaggtcctg 95 211516_at IL5RA ggcagccttccttgtgatcaaaaaaggtaatcccagaaacgtacccgttcactcgtg ggtcttaaaatggtttcatatctctattgtgactaattttctctcggtctactgccttttc aatcaggaatagatttgccatgaagccagtgaagtttttaagtgtctaggcttctcatt agtgccaactctcctagacctggtgcctgttttttttccaagttttgtttctacttctatcc attttttaaattaaactttttattttgaaataattatcacactcacaagctgtgggaaga aataatagagatcctgtgtctctttcatccagttttcctcaagggtaacatct 96 211772_x_at CHRNA3 tgctcaacgtgcactacagaaccccgacgacacacacaatgccctcatgggtgaag actgtattcttgaacctgctccccagggtcatgttcatgaccaggccaacaagcaacg agggcaacgctcagaagccgaggcccctctacggtgccgagctctcaaatctgaatt gcttcagccgcgcagagtccaaaggctgcaaggagggctacccctgccaggacggg atgtgtggttactgccaccaccgcaggataaaaatctccaatttcagtgctaacctca cgagaagctctagttctgaatctgttgatgctgtgctgtccctctctgctttgtcaccag aaatcaaagaagccatccaaagtgtcaagtatattgctgaaaatatgaaagcacaa aatgaagccaaagaggaacaaaaagcccaagagatccaacaattgaaacgaaaa gaaaagtccacagaaacatccgatcaagaacctgggctatgaatttccaatcttcaa caacctgtt 97 212359_s_at KIAA0913 cagcgctgccagcaggcatacatgcagtacatccaccaccgcttgattcacctgact cctgcggactacgacgactttgtgaatgcgatccggagtgcccgcagcgccttctgcc tgacgcccatgggcatgatgcagttcaacgacatcctacagaacctcaagcgcagc aaacagaccaaggagctgtggcagcgggtctcactcgagatggccaccttctccccc tgagtctttcacccttagggtcctatacagggacccaggcctgtggctatgggggccc ctcacacagggggagtgaaacttggctggacagatcatcctcactcagttccctggt agcacagactgacagctgctcttgggctatagcttggggccaagatgtctcacaccct agaagcctagggctgggggagacagccctgtctgggagggggcgttgggtggcctc tggtatttattt 98 212528_at — gtcactcatttccttgaacagcacccccctttatactagcagccatttgtgccattgcct gtgccctagggtttgtggggagagagcgagggatcactgagcagttttcccagagct ccatgggaaggcaagctctccctcccaatgggagccccactgtcactaactgtaaac tcaggctcaggcttcaactgcctacccccatcctcatatttctgtctgtcccagcacctc aggagcattctcattgtggccggctaactccgcctggatgtgaacaggcaagcacag tgggaaatgagtcacgtacttgtattgcacagtggacacctctagaggtccattggtt taaagggatagggaaggaggagggatgagaccatcaccccctcccagaagtaaat ctagtatctgagttttctttat 99 212531_at LCN2 caagagctacaatgtcacctccgtcctgtttaggaaaaagaagtgtgactactggat caggacttttgttccaggttgccagcccggcgagttcacgctgggcaacattaagagt taccctggattaacgagttacctcgtccgagtggtgagcaccaactacaaccagcat gctatggtgttcttcaagaaagtttctcaaaacagggagtacttcaagatcaccctct acgggagaaccaaggagctgacttcggaactaaaggagaacttcatccgcttctcc aaatctctgggcctccctgaaaaccacatcgtcttccctgtcccaatcgaccagtgtat cgacggctgagtgcacaggtgccgccagntgccgcaccagcccgaacaccattgag gga 100 213197_at ASTN1 tttccccttggaagacactattgatctcaacctgctgacttttcctaatgcttacctgaa ggaacccatcctggctagaaagggtgatggtactggaccggtattcaaccttgagttt tcaagctgccaaacaggtcttaagggaggtgcttatatcccaccaacactctcccag ctcccatgtccccaagacctctggagtttcctcttgaatgtacatgaaccactgtaata gcattagacttttaattgagtgtgcaatcgttttccatggagtttggtccgttcattattt tttagttaactacacttcttgatattcaaatgttctattaaaaaaactgagtatgaaga aaaacactttactactgcagaa 101 213260_at FOXC1 tcccccatttacaatccttcatgtattacatagaaggattgcttttttaaaaatatactg cgggttggaaagggatatttaatctttgngaaactattttagaaaatatgtttgtaga acaattatttttgaaaaagatttaaagcaataacaagaaggaaggcgagaggagca gaacattttggtctagggtggtttctttttaaaccattttttcttgttaatttacagttaa acctaggggacaatccggattggccctcccccttttgtaaataacccaggaaatgta ataaattcattatcttagggtgatctgccctgccaatcagactttggggagatggcga tttgattacagacgttcgggggggtggggggcttgcagtttgttttggagataataca gtttcctgctatctgccgctcctatctagaggcaacacttaagcagtaattgctgttgc ttgttgtca 102 213458_at FAM149B1 agcctgaaacaggaactcacatgagactcagggccaccaggaaatgcttaaaatac atactctttcccaaaagcaaatctataattctgtttcaattttatgaatatatgaatag acaaaatgaatcgaattacataactatgtcattcattaaatggcaacaatgctgaca gcaagcagtagatcctctgattccaattaccatttgttttttacccaattctatttgcta gaggtagtaagtactctggcactcataaatcacatgatgataaaaaggaacatgag gccgggtatggtggctcacaactgtaatccccataccttggg 103 213482_at DOCK3 tatgggtcagttacagcagccctcacctcaaagggctggcctgcttctcagcctacat tcatttgcaagcttcaatctctggaccatctggtgttcacaggtgttagagggttaggg gttaggggctagttttggatttgattcataggtaggagggcttagattttaaggcactt ctgaaagtcaatccctggacaaggcagtcatcacataagaacagctaccttctccac ttggtggcacaagaggtagggaggggagtatgggttcatttgncttcgcattatgca aggtgaaaccgtttgttttccctctccattttccctaactaaatgaaaaggacacattc tgaaatcccttttgttggagaataagtcagtctgaggggaaatgggaggccagagat gagaaccctttgaaaagattgtaaaatactgattttcattctttcaagcttatttgtaa atacctatttgaatgctgtgtatttgtacaggaatttgagcaaaaaatgtatagagtgt gatgtccaattggtattcagcactat 104 213603_s_at RAC2 gagcttcgttgatggtcttttctgtactggaggcctcctgaggcnnnnnnagcccca ggacccattaagccacccccgtgttcctgccgtcagtgccaactnnnnnatgtggaa gcatctacccgttcactccagtcccaccccacgcctgactcccctctggaaactgcag gccagatggttgctgccacaacttgtgtaccttcagggatggggctcttactccctcct gaggccagctgctctaatatcgatggtcctgcttgccagagagttcctctacccagca aaaatgagtgtctcagaagtgtgctcctctggcctcagttctcctcttttggaacaaca taaaacaaatttaattttctacgcctctggggatatctgctcagccaatggaaaatct gggttcaaccagcccctgccatttcttaagactttctgctccactcacaggatcctgag ctgcacttacctgtgagagtcttcaaacttttaaaccttgccagtcaggacttttgctat tgcaaatagaaaacccaactcaacctgctt 105 213917_at PAX8 ctgcctggttaccgtggcgatgtgcttaatgcagcgttgaaaatacagaatactgact cctctgtccctcctggccccggactccctccctccctcccttcctcttctggagcgtgaa atgagattggtcaagataaaaaaggaaaagattcggttatttttttaagagtgtggat aatggggcctctcaatcaaaatcccagtctccagtcggttccccccattccccttccaa cccctccaccttcccctgccgcctgcttagaggaggaggaagaaacataaagcaca aggcttttctcttaattatgaatcattccctgagggcaggcccagggcaaggggttcc tggggcccagagtctgacctgtgaggtagctagaaggcttgagcctctcatcaaagt cc 106 214457_at HOXA2 ctttgcaggactttagcgttttctccacagattcctgcctgcagctttcagatgcagttt cacccagtttgccaggttccctcgacagtcccgtagatatttcagctgacagcttaga cttttttacagacacactcaccacaatcgacttgcagcatctgaattactaaaaacat taaagcaaaacaaagcatcaccaaacaaaaactcctttgaccaggtggttttgcctt cttttatttgggagtttattttttattttcttcttgacctaccccttccctcctttaagtgtt gaggattttctgtttagtgattccctgacccagtttcaaacagagccatcttttacaga ttattttggagttttagttgttttaaacctaactcaacaaccctttatgtgattcctgaga gc 107 214608_s_at EYA1 gtcaccctgaggaaggttcattgccattgtcatcaccatggaaacaacgttcctctcc acctgcattatgtactacatgacaggcatcaatctggggaaataataaaattatcac ctttgtcagaccataagagtttctccaaaagtggtcagtttggctgggcaatatttnct ctcatctaacaaacacaatccattgtcatgaaattacccttaggatgagtcttctttaa tcaatcatatattgggcggaaaaaacaccagctttgacccgaagtagttgaagagct acttcattcttttctgaagttgtgtgttgctgctagaaatagtcatttgtgaattatcca aattgtttaaattcacaattgaattagttttttcttcctttttgcttgaagcaaacagttg acaatttttaaccttttcattttatgtttttgtactctgcagactgaaaagacaaagttt atcttggccttactgtataaaggtgtgctgtgtccaccgttgtgtacaga 108 214665_s_at CHP gaggtctggcactagtagcacaacctaaggtggcattacagatctttgagcgagcca cagcaacttttctgccaagtcagcttnagttnagacttcagtgaatcaggntattgct atcctaatgtatgtctctatgagtgtatntagccacanantctgcccttggttganttt ctgactcattgcttgcttgcttgtttccttgctttggaaaactatnnaagattgctaaaa aataccactgcaaagtgatggaaaagggtggagaacaggggagtagccaggctgg atggctcaaatataaatgaatgaggaattctttatgaagtatcagtcagattttatga ttaagtgatgtaatataggaattatgtaaaagggaagaatgtctgatactgatctatt agagaggtactttagaggcttcttgattggcataaagttcctaaggttatagattttcc ccccttttggctgtatagcaaagtgttttaatccacggttgtgccttattgttccattaa aa 109 214822_at FAM5B caatgggaggggtcggagctcttccttcccctctgtggagtcacttttgtattcttttta accagatttcttaaaatgttgttgttttgtgaatcctgacattggttcttacttttgtatg ctgcctcctctgtgccctcccagacgctgactgggaaacacaagaagtacaaccaac aggaaccagcgccaagggcaggcagcggcctccttgctcccctcccttactcctccct ctgctgcctcctccccccaccaagtttcagggccctggattgttcccagttcccattgtg gtcccttcagagctcctttccaacagcatctctctgtcgaagaaagaagctctgtcaa gttagagagagacaatgtgtaggaaatgttcttttttaaaaaaaaataacaaaaaca aaacaaaactatnnannntgtgattgttttccttgttaatctgctccaaccacctgaa catctaagta 110 215102_at DPY19L1P1 gagacgggagtttaccccgatcacagaaaccataccaactgaaagacaaatcagc atcttgctggacgacccctcacagagctcctagatccttgaagtgtgaacttcagcag ctgagagagatggggtctcactatgttgcccaggctggtcttgaactcctggactcaa gcaatcctctcacctcagcctcccaaagtgctgggattacagattttataaatattgtt gatctttttgaaaaaccaactgttggcttcattttntttattgtgtaatactaccttaga ggacagcagttcctaatacctacttttattatgagtctctgccatttataaagaactgt ggacagcacagggaatgggggaagaaaactctggtgcagcttgaatcttggtagca aaacagtgacttcatcagaaaattttgtcactctctattagatataatggagtttgacc atttggaatttggaatttttcaaatgaatatgacaaaaatttaaaaaactcttgtatta ctatgtgataacacagatctttacaacttta 111 215180_at — aagccttcaccagatggtcaagcagatgctggtgccatgcccttgancntcncncca ccatcccccacctagccactatatgggttgttagatattttgaccacctcctcttcnctc actccactattcaactcactgcatcatcaatgtacttattacaaacctgtcacaagcca ggtcttatgctaggtgctcctctcaacaggttcttgagctggcaggggagagagaga cattcaaacaccaaggattaatataccattacaggtttaaagacagaggcctataag ggtcccctggcagtgccatggaggtagggcatggtcggctgtacctgtagaggtgtct aaagggaggcttgcaagctgccccttgaaggacgagcagaaaattgtacatgagga caagtaggaaaggaattccaggaggagggatcagcatgtgca 112 215289_at HLA- ggactaaatcgagccttattatacatcagcagtctcacactggagaaagtccttttaa DRB1 gttaaggganngnnnnnnannntnnancaaatgtaatactggtcagcgccaaa /// HLA- aaactcacactggagaaaggtcttatgagtgtggtgaatccagcaaagtgtttaaat DRB2 acaactccagcctcattaaacatcagataattcatactggaaaaaggccttagtgga /// HLA- gtgaatgcaggaaagtcaccaaaactgtcacctcattcagcaccaaaaggttcacat DRB3 cggaccaagaacctattaatatatgtaaatctaatgttgaaagagttcagatggaaa /// HLA- tctgcgaggatttcctgctgggaactacatta DRB4 /// HLA- DRB5 /// LOC100133484 /// LOC100133661 /// LOC100133811 /// LOC730415 /// RNASE2 /// ZNF749 113 215356_at TDRD12 aattgggcaggctcttgggaagtagaaagttctggtgtttttgctggtgaaggttttga ctgtggagctcttctaacacccatatcagtgtctgtttctctgcatgtggctgctgccct gttggtggagctctgggggcagagaccaggccgccgtccagtggcgcnccgtgcgc accagctgcctgctgtttacacccaggtgcgccgagtctctttcatacagcacagcaa atgataatagctagtgacaatgtgtttcctgtgcactcgtgaaaatgcagggaggac aactgcatgcttagatctgtttcttttttcagacattcaaatgttctaatatctgaagct aacattttgtaggatataggatgctgattatgtgaacaattagtcattggttttctgtac tgctatgaatatgtctgatttcaagttttggtcaaatatctaaaatgcaaggtgaaagt gcctttgtctctatgcttctaaaatcgctcatgcttagttgtggtatggatgtcttccgc agtg 114 215476_at — cttggtaagccttgcctgtagcggctccgctgccgagtgctttgacaccaggcgctcc cagagctctgcccccactgccaagcggcagctgctccggagggcacggggggctgg atttggctgtggcttctccagctctgcacaagagccccccttccctggccctgctgcag catgactgcctcctggctcgtgtcacccactctgtctctgtctctcttcatacgtttccag ctgagctgggatccatagtctgtttccctctccacgaccaatctatttatcttctctgga acttcttgtaatgccgggagtgcagagcttacaagttggggcaggaagctttagaag cccaggnagccctgagaggctctttccttgtaagtgggtctctccccaggagcctctt ggaatatttagcagggacttttacccatgctgggtctagagaccctcccgcccctctgt ttcctgccctcctacttagactgggatctggtttccctcagctggttcccttgctagcgt gtgactctgtgtgtct 115 215705_at PPP5C gttcacagcagtgggtaggcccagcagtggttcttgacatcacacgatgaggcgngc atctcccgtcatccagggagaccagaggacccttgtctcactcccagttggctnttag tcacagccccgctttgtctttgacatggacgtttgtgatgatcacgttcctcccgctccc cgtgtntgaagagtgctccctgactggctgccgtctcctccctgtcgggtctggctggg ttctccanagggagtgctgcggaggggacacagcanaggccccatgctcgtgatgt atgttgcagatcattttcccccattctgtccttttttgttaaattgtggtaaaaagcaca taacataaactgtaccnccttaaccatttgaaagtatatatcccagactgtcttttatc tttagacttcacttgtggtttgttgcc 116 215715_at SLC6A2 tcccctggaagttgtcctttctgatcctctcttcttttcccatttacaaatgatttcgtga ctgtagtttttgttcaccttctgtgcatctggcctgggggctgttagctcagaggagag gagcaaacaggaaaatgacttctgttctgtccccgctgttttgggggaagtctctccc actttgggatcctgctgaagctaggttcatgaggtcggaaatccccaccacatttgcc tagactttgggcacaggagttcttagtccaccaaatcaga 117 215850_s_at NDUFA5 cattttctctaactttatctcctatgcatttccttatgtgtcctgtacagcagtatattcc aaaatccccagtggatgtctgaaaaccacatatagtaccaaactgtatatatgctat gttttgtttcatacatacctataataaagtttaatttatgaattaggcacaataagaga taagcaggctggacgtgctggctcacgcctgtaatcccagcactttgggaggctgag gcgggtggattgctttagcccaggagtttaagaccagcctggccaacatggcaaaac cccgtctctataaaaaatgtggaaattaatcaggtgtggt 118 215944_at — gagatgaccgaaaacttcaacccctgcagtcagcaatggtcaacagaaagggccca attctccacgacaatgcatgatcgcacattacacaactaaagcttcaaaagttgaac taactgggctacgaagttttgcctcatccaccatattcacctgacctcccgccaaccg actaccacttcttcaatcatctcgacaactttttgcaaggaaaacacttccacaacca gtagaatgcaaaaagtgctttccaagagttcactgaatcctgaagcacggatttttat gctacaggaataaacaaacttatttttcattggtaaaaatgtgttgattgtaatggatc ctattttgattaatgaagatgtgtttgagcctagttataatgatttaaaattcacgatcc aaaaccgcaattacttttgcatcagcctaatatgaggaagtaatagttgaacagaat aattctttcctggaagtct 119 215953_at DKFZP564C196 ttggtttggtctggtttggctacctgattcctgctgtctttttctacgccaggtgaagag gcactttcaagatccttctctgagacctgcaccaataagactataccaatgttcagttg aaacatcaggtataagtttagcggaaacgaaagtacaacctgctttgaaataaattc caaggacagattgtcattaacgaaatagaaagtggactatgcccctcatgctgccag cgcctggtatgatgcggcgtgacacgcagcgcttgcggcagtacaatgcccccaatc acccgccccgccccgacgcgccgcccactcacggcaaagagagccacctagtgagg gattattctcatttccgcggtggggttctgcttttctttctaccatgagcgcccaaggat agacactcctactacctattacctcaaatagcctacatttctttccgaa 120 215973_at HCG4P6 agaacactgagcgaggctctgtagatggatgtaataaaaatctataaaacaatgtgt ttaaacctaagaattctactgctttccaattccttccctctgctccttttcctaacctcct gcttctccagcccttccctctgtccctttcanccctcaggccctcctctccccttagtccc caccaccctgtcacttctaaattgtggctctagcattgtcccattacctgctangtgac tgttctctccacagtggtcctgctcctgtgagtcagagtgtgtcatttcctcacctaaaa cactccagtggctccacctcggtcttgtgaagcttctagaatgtcaggcacgtgagca tatgagggcatacctggttcatcttaggcactaaattnnnntttgttgactgaatgaa tgaaatatgaatgtattaaattgcatcacagaaagttataaaatgtaaaacactgaa aaattaagaaatattttatnttatgtaactagtgtgcatatcaattcattccgagtctg ttgagcctgtgtat 121 216050_at — aatgattcaactcatgtgatccagtgttacattcagtgtggtaatgaagaacagtcaa aacaggcttttgaagaattgggagataatttggttgaattaagtaaagccaaatact ccagaaatattttaaagaaatgtctcacgttgtgaacatgtaccctagaacttaaagt ataataaaaaaaaaaaaaannggaaagtatcttgcacaagctcacgtagctggta agttacatagttgggatctgaattcagttgtggcttcatgcctgagcttttaactactac tactaaactgagaaggcacttgcttgagtaaattatgtcatcctcttaat 122 216066_at ABCA1 gatgtggcatgtgatgacattgcacatggncagttaantgngccaagaagngcagc agtagcagcaacnggagatgcaaagcccaacatgatggggagagaaantnttctt tcaatatgtgcttctgtaccaaaagtggaatttcacgagagacatattttggaacattt ttccttttgtgtgtgcgtgagtgtttccctgtttccagccaagggtattgtgagtttctcc tgggcctccttcagaatctgggtgctctggaaagcagtgttttggcaacatggggaaa gtatggcagtgtgggagggtcagctgggtctgggtttgaatattgcatttgaatatttt accagcattgatgtcggataaattatttagtccctgtaagcctcagttttntcttnttct acatacacataatatatttgactctttgttgtgat 123 216240_at PVT1 tttcctaactttctgatcccttggaggtgataatcaaatattctagtctgaggcattggg atacatggtgctaggttctgagactctgcgtcaggcctgaaccctgcattttgtggag gtgggtgggagaatgtncccctggggaacatgcctagacacgggggacaacagttg ccctcatggggaggtacctgtttactcgctgttatgggaccgctttcacaaaaccact gcaggtgagtgagttcctgctgaatatcaggcctggtgtctctagactcattattnccc ccacccaacccctatgttagttcatctcgagccacatttttattgccataatccaggcc tggacaggccaagatcttttaacaattttaattactgaaaataataactgcatttttttt naaagcccaacttttnggtanagtcagcccaaaatacagtctttgtgttgccatctgg gaactggatttggaattgttcttccatgagactgcagagcag 124 216881_x_at PRB1 ccacctcctccaggaaagccagaaagaccacccccacaaggaggtaaccagtccc /// aaggtcccccacctcatccaggaaagccagaaggaccacccccacaggaaggaaa PRB4 caagtcccgaagtgcccgatctcctccaggaaagccacaaggaccaccccaacaag /// aaggcaacaagcctcaaggtcccccacctcctggaaagccacaaggcccaccccca PRH1 gcaggaggcaatccccagcagcctcaggcacctcctgctggaaagccccaggggcc /// acctccacctcctcaagggggcaggccacccagacctgcccagggacaacagcctc PRH2 cccagtaatctaggattcaatgacaggaagtgaataagaagatatcagtgaattca /// aataattcaattgctacaaatgccgtgacattggaacaaggtcatcatagctctaac PRR4 125 216989_at SPAM1 gtttgatgtctattatctcacttcatcctcaccaggaccccatccgagccttaatttcag ttgacagtaactattggatccccaggaatatgtttgcatatttggggagaaaatacta ttggaggggaacagaaatgctactaagggtctcactgtgtcacccaggctggagtcc atcaaagctcactgcagccttaaccttctgtgctcaagggatcctcccacttaagcctc ctgagtagctggaactacaggcatatgccaccgagcctggctaatctttgatttttttg tacagattgtgtctccttatgttgctcaggctggactcaaacttctggtctcaagcgat ctttccatcttagcttcccaaattgttggaattatggacatgagccagtgtgcttggcct gattttttttttttttttaatgagaaaaacgttccttaagaaaagtttcattgtaagacg aggacttgctatgttgccagtttggtcttgaactcggtctcaagtgattctcctgccttg ggttcccaaagcgtttgggccggcagatgt 126 217004_s_at MCF2 ctgaattggaacacaccagcactgtggtggaggtctgtgaggcaattgcgtcagttc aggcagaagcaaatacagtttggactgaggcatcacaatctgcagaaatctctgaa gaacctgcggaatggtcaagcaactatttctaccctacttatgatgaaaatgaagaa gaaaataggcccctcatgagacctgtgtcggagatggctctcctatattgatgaagct actatgtcaaatggcaagtagctctttcctgcctgcttctcagctcatttggaaaaata ctgcgcaaaagacattgagctcaaatgatgcagatgttgttttcaggttaatggacac gcaaagaaaccacagcacatacttcttttctttcatttaataaagcttttaattatggt acgctgtctttttaaaatcatgtatttaatgtgtcagatattgtgcttgaaagattctca tctcagaatacttttggact 127 217253_at SH3BP2 gagtgtcttgactattctggctctttgtattttcatgtaaggtttttctcccatataagttt taaaatcagcttgtcaattccaacaacaatgatgcacttgatagtttgggaatttatta tagctatcaatcagttttgggaaaattgacgtctttacaatattgagttttctgattcat gaacatggtttacctctcttcccatgggggtctcctttaaggtttaccaataggatttta tatttggggccattgnggtcttgcttatcttaagtnnnnnnnnnnnnnnnaaatct cttgaccncatgatctgcccgccttgtcctcccaaagtgctgggattacaggcgtgag ccaccgcacctggcctgcaatacagtattgttaaccgtcttcaccatgttgtacgttag agctccagaaattatttancatgcataactgaaactttatactctttgaacaccacctc cccatttccctctcccggcagccatttgtgcctctcggttctctttattagcttccattttg tgggtcagt 128 217995_at SQRDL tacgtcaaagaccgctgctgcagtagctgcccagtcaggaatacttgataggacaat ttctgtaattatgaagaatcaaacaccaacaaagaagtatgatggctacacatcatg tccactggtgaccggctacaaccgtgtgattcttgctgagtttgactacaaagcagag ccgctagaaaccttcccctttgatcaaagcaaagagcgcctttccatgtatctcatga aagctgacctgatgcctttcctgtattggaatatgatgctaaggggttactggggagg accagcgtttctgcgcaagttgtttcatctaggtatgagttaaggatggctcagcactt gctcatcttggatggcttctgggccaaaactgcagtcactgaatgaccaagagcagc acgaaggacttggaacctatccttgtaaagagttccttgatgggtaatggtgaccaa atgcctcccttttcagtacctttgaacagcaaccatgtgggctactcatgatgggcttg at 129 218768_at NUP107 ttggatgccctaactgctgatgtgaaggagaaaatgtataacgtcttgttgtttgttga tggagggtggatggtggatgttagagaggatgccaaagaagaccatgaaagaaca catcaaatggtcttactgagaaagctttgtctgccaatgttgtgttttctgcttcatacg atattgcacagtactggtcagtatcaggaatgcctacagttagcagatatggtatcct ctgagcgccacaaactgtacctggtattttctaaggaagagctaaggaagttgctgc agaagctcagagagtcctctctaatgctcctagaccagggacttgacccattagggt atgaaattcagttatagtttaatctttgtaatctcactaattttcatgataaatgaagtt tttaataaaatatacttgttattagtaattttttcttttgcattaccatgtaaaatttaga catttgaattttgtacttttcagaatattatcgtgacactttcaacatgtagggatatca gcgtttctctgtgtgct 130 218881_s_at FOSL2 aggtcacagtatcctcgtttgaaagataattaagatcccccgtggagaaagcagtga cacattcacacagctgttccctcgcatgttatttcatgaacatgacctgttttcgtgcac tagacacacagagtggaacagccgtatgcttaaagtacatgggccagtgggactgg aagtgacctgtacaagtgatgcagaaaggagggtttcaaagaaaaaggattttgttt aaaatactttaaaaatgttatttcctgcatcccttggctgtgatgcccctctcccgattt cccaggggctctgggagggacccttctaagaagattgggcagttgggtttctggcttg agatgaatccaagcagcagaatgagccaggagtagcaggagatgggcaaagaaa actggggtgcactcagctctcacaggggtaatca 131 218980_at FHOD3 gcacctcggagttgcagctgtgacactcataggttactcccaggagtgtgctgagca gaaggcaagctcttgctggatgaaacccctccaggtggggttggggagacttgatat tcacatccaacagtttgaaaagggagagctcaattcccagcgtcaccccatggcttgt gttgcctgctacgcattgacttggatctccaggagtcccctgcacataccttctccatc gtgtcagctgtgtttctcttgattccgtgacacccggtttattagttcaaaagtgtgaca ccttttctgggcaaggaacagcccctttaaggagcaaatcacttctgtcacagttatt atggtaatatgaggcaatctgattagcttcacagactgagtctccacaacacc 132 219000_s_at DSCC1 tcaagtgagtgagttcccctctacttttagccttccacccaaactggaagcctctaggt gctatcaattatttatatccatcgtttacatccatgaaattggctgaataattactcctc tgcctggcgtagacatgtgctttgggaaaaaaacgagtttataatcctataatgaag aatactggcacaggcaatgctcactcgaaaacttcaagtaatttctagttggttttgg aatgcttgataaagttcctttacagctttattttcctgatttgttttggtttagatcaaag ttcaaattaattttaacttagctaatgaactcatcaccaggacagttggagggggtag gccgaggttaaatggtccacgtttcaaaaatgttaat 133 219171_s_at ZNF236 cttttgttcttgctgggttatttattttgattttagcattaaatgtcatctcaggatatctc taaaaggggttgtttaattcctaattgtatagaaagctagtttggtgaattgtattggtt aattgactgtttaaggccttaacaggtgaatctagagcctacttttattttggttaaag aaaaagaaaatatcaataattcaattttgtgtcttttctcaatttattagcaaacacaa gacattttatgtattatttcgatttacttcctaattataaaagctgcttttttgcagaaca ttccttgaaaatataaggttttgaaaagacataattttacttgaatctttgtggggtac aggttgatctttatattttactggttgttttaaaaattctagaaaagagatttctaggcc tcatgtataaccagggttttgaggataaagaactgtatttttagaactatctcatcata gcatatctgctttggaataactat 134 219182_at FLJ22167 ttaccctcgtggctaagcaagtgtctgcaggagcagagatggctggaaggggcctct gcacacggaagatggcttgttcagcccattcacctcctgaggatgtgggcagtctcct ccaagaacacatggagctgcttcctgatcccaagcaggtcattgccactggaaggac atggccccggtgatccatgcttcatgcccacccagaaacacacccctcagtgtgtgcc tcagtttactttggagatcagttgtcgtttttagtgctcctttaggcttactaaaacagtt ttggaaacaaagctattttgaagtattcaagcagaggaattccctaacactgacc 135 219425_at SULT4A1 gaccattttgcgagtgtagccctgtttcactcggatcaggttggcacggccgcctgcgt gtctgtccacctcatccctccgtgtatctgagggagtaaaggtgaggtctttattgctt cactgcctaattttctcacccacattcgctgaagcgatggagagtcgggggccagta gccagccaaccccgtggggaccggggttgtctgtcatttatgtggctggaaagcacc caaagtggtggtcaggagggtcgctgctgtggaaggggtctccgttcttggtgctgta tttgaaacgggtgtagagagaagcttgtgtttttgtttgtaatggggagaagcgtggc caggcagtggcacgtggcatcgcatggtgggctcggcagcaccttgcctgtgtttctg tgagggaggctgctttctgtgaaatttctttatatttttctatttttagtactgtatggat gttactgagcactacacatgatccttctgtgcttgcttg 136 219520_s_at WWC3 aaggaaggccagagagccgcgcagttctctgcaggtgcagatgcaggcagtggag gtggcctgagcaggcagaaggacaccaagcgccctatgttgcttgtcattcatgacg tggtcttggagcttctgactagttcagactgccacgccaaccccagaaaataccccac atgccagaaaagtgaagtcctaggtgtttccatctatgtttcaatctgtccatctacca ggcctcgcgataaaaacaaaacaaaaaaacgctgccaggttttagaagcagttctg gtctcaaaaccatcaggatcctgccaccagggttcttttgaaatagtaccacatgtaa aagggaatttggctttcacttcatctaatcactga 137 219537_x_at DLL3 tcccggctacatgggagcgcggtgtgagttcccagtgcaccccgacggcgcaagcg ccttgcccgcggccccgccgggcctcaggcccggggaccctcagcgctaccttttgcc tccggctctgggactgctcgtggccgcgggcgtggccggcgctgcgctcttgctggtc cacgtgcgccgccgtggccactcccaggatgctgggtctcgcttgctggctgggaccc cggagccgtcagtccacgcactcccggatgcactcaacaacctaaggacgcaggag ggttccggggatggtccgagctcgtccgtagattggaatcgccctgaagatgtagac cctcaagggatttatgtcatatctgctccttccatctacgctcgggaggtagcgacgcc ccttttccccccgctacacactgggcgcgctgggcagaggcagcacctgctttttccct acccttcctcgattctgtccgtgaaatgaattgggtagagtctctggaaggttttaagc ccattttcagttctaacttactttcatcctattttgcatccc 138 219617_at C2orf34 tgaagaaaaccttcattacccgcttctgcttattttgaccaaacatggatagaagatt aagcttctcaaagacgaagaaacgtatcaagtgcatagggaatatttttacaaaaac ggaaatctgtaaggggtataatcgcctgcctgcgccctttgcagcatttcacgtgtgg gctatggactccacctgtcctcacccacgttattccccagctgccctctccagctccct ccccgcctctttttacactctgcttgttgctcgtcctgccctaaacctttgtttgtctttaa atgtgtataagctgcctgtctgtgacttgaatttgactggtgaacaaactaaatatttt tccctgtaattgagacagaatttcttttgatgatacccatccctccttcatttttttttttt ttttggtctttgttctgttttggtggtggtagtttttaatcagtaaacccagcaaatatca tgattctttcctggttagaaaaataaataaagtgtatctttttatctccctc 139 219643_at LRP1B tattcacaagttttggagggctttttgttcctctgatagacatgactgacttttagctgt cataatgtattaacctaacagatgaaatatgttaaatatgtggttgctctttatcccttt gtacaagcattaaaaaaactgctgttttataagaagactttttgttgtactatgtgcat gcatactacctatttctaaactttgccatattgaggcctttataaactattgatttatgt aatactagtgcaattttgcttgaacaatgttatgcatatcataaactttttcaggttctt gtttaagtacattttttaaattgaacagtatttttcattttggttataatatagtcattttg cctatgtttc 140 219704_at YBX2 ctcagcccctgtcaacagtggggaccccaccaccaccatcctggagtgattccaact caactcaaaggacacccagagctgccatctggtatctgccagtttttccaaatgacct gtaccctacccagtaccctgctccccctttcccataattcatgacatcaaaacaccag cttttcaccttttccttgagactcaggaggaccaaagcagcagccttttgctttttctttt ttcttccctccccttatcaagggttgaaggaagggagccatccttactgttcagagac agcaactccctcccgtaactcaggctgagaag 141 219882_at TTLL7 gtttctgtgattcaggatcctcttgggagagtatattcaataaaagcccggaggtggt gactcctttgcagctccagtgttgccagcgcctagtggagctttgtaaacagtgcctg ctagtggtttacaaatatgcaactgacaaaagaggatcactttcaggcattggtcctg actggggtaattccaggtatttactaccagggagcacccaattcttcttgagaacacc aacctacaacttgaagtacaattcacctggaatgactcgctccaatgttttgtttacat ccagatatggccatctgtgaaacagaagggaagatcgccattggttat 142 219937_at TRHDE ggaggtcccaaatatgtggtctatcaccactgaattcatgtaatagataagaaaaaa attagaggtggatgtcttgttttgtgtcatgaattactaaaatctcttagtagttgtggt atatttttgagtaaaattaccatttccagatttgagtttgaagggcttttatagttgtatt ttcctcctcactgttaataatcataatcctttttcagtattttagtggccttgaacaactg gtttatctacaatctcaaatcctaagtgtataattatgtgcaatgttcaatacctcatat aatacttgctcaacagtatagtggtaccaatggcattaagatggtgtttttgttctaca tatttttcaataatttattctttctaatgttgaaattatatcaggctttaccggtt 143 219955_at L1TD1 gaagttgcaacattcgtttgataggaattccagaaaaggagagttatgagaatagg gcagaggacataattaaagaaataattgatgaaaactttgcagaactaaagaaag gttcaagtcttgagattgtcagtgcttgtcgagtacctagtaaaattgatgaaaagag actgactcctagacacatcttggtgaaattttggaattctagtgataaagagaaaata ataagggcttctagagagagaagagaaattacctaccaaggaacaagaatcaggtt gacagcagacttatcactggacacactggatgctagaagtaaatggagcaatgtctt caaagttctgctggaaaaaggctttaatcctagaatcctatatccagccaaaatggc atttgattttaggggcaaaacaaaggtatttcttagtattgaagaatttagagattatg ttttgcatatgcccaccttgagagaattactggggaataatataccttagcacgccag ggtgactaca 144 220029_at ELOVL2 gttatacagatgccatgctccacaccacgagcagtgtacaaatctggctgcccgttta ctttctgagcaagcactggagtccactccgacctttttctttgaacatgcatgctgctg gaatatgtataaatcagaactagcagaagtagcagagtgatgggagcaaaatagg cactgaattcgtcaactcttttttgtgagcctacttgtgaatattacctcagatacctgt tgtcactcttcacaggttatttaagttcttgaagctgggaggaaaaagatggagtagc ttggaaagattccagcactgagccgtgagccggtcatgagccacgataaaaaatgc cagtttggcaaactcagcactcctgttccctgctcaggtatatgcgatctctactgaga agcaagcacaaaagtagaccaaagtattaatgagtatttcctttctccataagtgca ggactgttactcactactaaactct 145 220076_at ANKH gaacgtcgtatgagatcctacaatggaagaataaaatcacctcattcttcatttcaga tctgaacattagcagtgatctagatttttttttttttaaacaaaattaagtgtgcttaga gtcatccctctacatgggctgtggctgtcagcccataggtttgtcagtttcacatcaaa actgtgggtataaactgttgaaaccaatcacattaaaatatttagctgggcacagtg gtgtgcatctgtagtcccagctacttgggaggctgaggcaggaggatcgcttaagca caggagttggaatccagcctgagcaacagagcaaaaccccgtctctaaaatacaaa taaaatatttgtgtagtttttgattaaaattgactacagcggtcagtataaaatacatg tcgcttttaaggaagtgctctttatgtatctaacagatggaagtttttgcattggtaag agcatttatatatgctttgtttcagggtttatggatttgtattcatatattgtcaaatagg tttcatactctaattttactt 146 220294_at KCNV1 agattatatccctatcttctttttcatgtaaaccactggtcacaaatgaactgatctctg tatcccattattactataagaggtgggaatcccaaaactgcttagattgcagtacatg agtttacacaaagacttcaacaattgcacatcttcattctcccaactgagtgtagtatg tggagcataaaacagcatattcttagtatttcatgaatatcagatggtctttaaatgtc tctttatggatgtattgttcacattatggctttaaaataatgaatatgtaaaagtgagg tagtgaacatcctaaatttctacactggaattactaaataatcttatttcataaaatgg gaaatatatgttaaatgacatcactggatgaacttgaagatcttttacttgttaacaa aaaaatactatggacagctttctgattgttggggtaaatagcaaatgttcaaactttg caggcattttgacattcatcataacaacacaattcctagacatt 147 220366_at ELSPBP1 ttaggcagtctgtggtgctcagtcacctctgtcttcgatgagaaacagcagtggaaat tctgtgaaacgaatgagtatgggggaaattctctcaggaagccctgcatcttcccctc catctacagaaataatgtggtctctgattgcatggaggatgaaagcaacaagctctg gtgcccaaccacagagaacatggataaggatggaaagtggagtttctgtgccgaca ccagaatttccgcgttggtccctggctttccttgtcactttccgttcaactataaaaaca agaattattttaactgcactaacaaaggatcaaaggagaaccttgtgtggtgtgcaa cttcttacaactacgaccaagaccacacctgggtgtattgctgatgctgaggaaagg agaaatatcttcagaggaagactgccgccatactgaggctgagcacagatttgtcttt ttcattgcatctgtcaa 148 220394_at FGF20 gtgtggcagtgggactggtcagtattagaggtgtggacagtggtctctatcttggaat gaatgacaaaggagaactctatggatcagagaaacttacttccgaatgcatctttag ggagcagtttgaagagaactggtataacacctattcatctaacatatataaacatgg agacactggccgcaggtattttgtggcacttaacaaagacggaactccaagagatg gcgccaggtccaagaggcatcagaaatttacacatttcttacctagaccagtggatc cagaaagagttccagaattgtacaaggacctactgatgtacacttgaagtgcgatag tgacattatggaagagtcaaaccacaaccattctttcttgtcatagttcccatcataaa ataatgacccaagcagacgttcaaa 149 220397_at MDM1 tatgcattttttaccacaatttttaaaaagtttgaatagaaatttttaatgtctttgagtg gattttgttttttgaacagttggatagacttctgcgtaagaaagctggattgactgttgt tccttcatataatgccttgagaaattctgaatatcaaaggcagtttgtttggaagactt ctaaagaaactgctccagcttttgcagccaatcaggtagcttaatggatgtaatacat ttctgagtaccattatcttatctagtaatgtagatttacatagaattaagagttgaaag aaattaagtacttaagtagcctggaggtaggttctagaaaaccaaaatgagagtttt gctaaaatcatcctattacttatgatttatggtagtaatattatactgtcctaggcttct gatgatcattgttgccagatgcagcacatatactaaatatgagacagggtaatgaaa acttggggaactggtaagtttttgcatgctac 150 220541_at MMP26 tgacccctttgatattccagcaagtgcagaatggagatgcagacatcaaggtttcttt ctggcagtgggcccatgaagatggttggccctttgatgggccaggtggtatcttaggc catgcctttttaccaaattctggaaatcctggagttgtccattttgacaagaatgaaca ctggtcagcttcagacactggatataatctgttcctggttgcaactcatgagattgggc attctttgggcctgcagcactctgggaatcagagctccataatgtaccccacttactg gtatcacgaccctagaaccttccagctcagtgccgatgatatccaaaggatccagca tttgtatggagaaaaatgttcatctgacataccttaatgttagcacagaggacttattc aacctgtcctttcagggagtttattggaggatcaaagaactgaaagcactagagcag ccttggggactgctaggatgaagccctaaagaatgcaacctagtcaggttagctgaa ccgacactcaaaacgctac 151 220653_at PEG3 aaggtagaaagccttccgtccagtgtgcgaatctctgtgaacgtgtaagaattcaca /// ZIM2 gtcaggaggactactttgaatgttttcagtgcggcaaagcttttctccagaatgtgcat cttcttcaacatctcaaagcccatgaggcagcaagagtccttcctcctgggttgtccc acagcaagacatacttaattcgttatcagcggaaacatgactacgttggagagaga gcctgccagtgttgtgactgtggcagagtcttcagtcggaattcatatctcattcagca ttatagaactcacactcaagagaggccttaccagtgtcagctatgtgggaaatgtttc ggccgaccctcatacctcactcaacattatcaactccattctcaagagaaaactgttg agtgcgatcactgttgagaaacctttagtcacagcacacacttttctcaacattattgg cttcctcctagagtgttgtgagtgtgagaaggcctttcactagcccc 152 220700_at — atgttactacaaacttgattaaacttctggtggaaattccatcacattttatgcaatttt caatttatttctccaatttatttttaatgccacatggacattatattccttaaccattcttt tgcatgtgattaacatttgtgaaattaaccacttaagcaagtgtttttgctttgatgaa agaaaaatgtttaaaatcctactggatatgaaactgaaagtaatgttttgtgttttttg tttcaaatgaaagtgtaaattaagaatttgttggcagggcgtggtggctcatgcctgt aatcccagcactttgggaggccgaggtgggcagatcacctgaggtcagcagtccaa gaccaccctggccaacatggtgaagtcccgtctctactaaaaatacaaaaatcagct gggcatggtggcgggcacttgtagtcccagctactcaggaggctgaagcaggagaa tcacttgaactcaggaggcagaagttgcggttagccga 153 220703_at C10orf110 cctctctccactctctagaaatattaaggctaggctgctgctgtatgtcagggctagtc ccctcttctatgaatccagaataactctgaagaagccgagtaacaggcatgaagtga agagaaatcgctgtaacaggaaga cagcaaagcagatgctaatgaccacactattt aacgaactggaaccaacgagaaaatacggtattactgaagactgcacttccttgaa cagagtgctcttctcagcaaatcggaaatgcctacacaaatcgctttacaagaaaga ctgtttcaaagcagcacctttctcaatgttctcgttcaggtgacaattcttcttggtctc agctccaattttattgtcattttcatcaataaggatacacatctctgccaggagttgaa cctgttgcttgtcgaggtggttagtgtttatttcaggcatcattacaaaatgtctgatct gttctagaaccct 154 220771_at LOC51152 aagtatctccatacaaaatacggttgaattacaaaaagaaaattgtaacattagcat ggacaaacctggcaggtactccttaactctcctaagtaataaaaactgtaaaatgca aataagccttcgatgacatttactaacctttactaaagtatcaatgatgacttggttgt ttaaacagctgacatttgggcaatttgagtatgtcaaactcaataatactggttttcat ttgcaagatccacttaaaacttaaggaggccaaaaaacatcatttaaaataccctat aaattataatcatacatatgatacgaaaaatatcctacttcag 155 220817_at TRPC4 catacacatacgtattttccgtagtgctctgggtgggggaaaatgtttaaattgtatta gcaaatgctaacttacactttatagcatttatcagctgtggcatattacctgtaacatg tttaaattaaggcaaaggcaatcaaaaacctttttgttttgtagcctgcttttgctttca caatttgtcttacaatt 156 220834_at MS4A12 gctggccaagactactgggccgtgctttctggaaaaggcatttcagccacgctgatg atcttctccctcttggagttcttcgtagcttgtgccacagcccattttgccaaccaagca aacaccacaaccaatatgtctgtcctggttattccaaatatgtatgaaagcaaccctg tgacaccagcgtcttcttcagctcctcccagatgcaacaactactcagctaatgcccc taaatagtaaaagaaaaaggggtatcagtctaatctcatggagaaaaactacttgc aaaaacttcttaagaagatgtcttttattgtctacaatgatttctagtctttaaaaactg tgtttgagatttgtttttaggttggtcgctaatgatggctgtatctcccttcactgtctctt cctacattaccactactacatgctggcaaaggtgaaggatcagaggactgaaaaat gattctgcaactctcttaaa 157 220847_x_at ZNF221 tgacatgcaccagagggtccacaggggagagcgaccctataattgtaaggaatgtg gaaagagctttggctgggcttcatgtcttttgaaacatcagagactccacagtggag aaaagccattgaaatctggagtgtgggaagagatctactcagaattcacagcttcat ttacatcagtaagtctatgtgggagaaaagccatataaatgtgagaagtgtgggaa gggctttggctgggcctcaactcatctgacccatcaattctccacagcagagaaaaa ccattcaaatatgagaactgtgggaagagctttgtacatagatcatatcttttttttttt ttttgagacagagtctcactctttcacccaagcctgactgcagtggcg 158 220852_at PRO1768 gaaaagcgccctgtgctgagtaaagcagccagtcttctcttgtcacagtaaaaggct gggagtaaaatttcccataaacacaggggaaacctacatttactcacatgccaagg aaaatggcacggaagacccacgtgtagccacagcagagtctatgcagagggcctgc aaatgcctggggtgcgagtgaatgcctggaggggcggagtttccaagataacagct attgtgttttctttttcacacttcagaagagaatcctaaggactagactccgctcagtg cattcctttttcatacactgatctcaagtacaatcacataattttgaaaatccatgtagt cctccctaaataaaattataaggataggtttctatttccttccgattacctagatacctc cgtcttctggaaaaccccaaaaagaccagtagacgaatcaggaaggtcctaggagt gattcctccaat 159 220970_s_at KAP2.1B tgcccccacagagcaatacactgaagcctaaacatctatctggtgtttttaaaaagtt /// aaaagaaaaatagattttttttcacaaggtgacaatagtgatttttaccatctggata KRTAP2- cagcctggtgtaagcagacgtccattaccaccctcacccacattttcaggtgtctaca 4 /// tcagccttagtcattatggatagtaaatcgacctttaagaattcctggggtggactttg LOC644350 caaacacattctacaacctgatggtttttactgctcaaactgtcaccatcatcttttgca /// atgtgttgctcactgttgtcaata LOC728285 /// LOC728934 /// LOC730755 160 220981_x_at LOC650686 ggacagtctcagggttctgttctcgccttcacccggaccttcattgctacccctggcag /// cagttccagtctgtgcatcgtgaatgacgagctgtttgtgagggatgccagcccccaa NXF2 gagactcagagtgccttctccatcccagtgtccacactctcctccagctctgagccctc /// cctctcccaggagcagcaggaaatggtgcaggctttctctgcccagtctgggatgaa NXF2B actggagtggtctcagaagtgccttcaggacaatgagtggaactacactagagctgg ccaggccttcactatgctccagaccgagggcaagatccccgcagaggccttcaagca aatctcctaaaaggagccctccgatgtcttctttgtcttcgttcacatcctctttgtttcc tcttttcaccagcctaaggcctggctgaccaggaagccaacgttaacttgcaggcca cgtgacataac 161 220993_s_at GPR63 aagtctgcattgaatccgctgatctactactggaggattaagaaattccatgatgctt gcctggacatgatgcctaagtccttcaagtttttgccgcagctccctggtcacacaaa gcgacggatacgtcctagtgctgtctatgtgtgtggggaacatcggacggtggtgtg aatattggaactggctgacattttgggtgatgcttgttctttattgacattgaattctctt tctcatagcctctccactttatttttttttatagggtttgtgtatgtatgtgtgtgagcagt gtaaagaaagaatggtaattatagttctgttaccaagaataaataataggaaagtg attacaaatattacctccagggttcaatagaaatcctcaatttagggtgaggagactt ttttttggttttggggtttttccttgattgattttgttttcatagtgggaatcaggattgtg ctttattgagcctgcagttacattgaattgtaggtgtttcgtgtgctgctaaggta 162 221018_s_at TDRD1 gggactgtcgatgtagctgataagctagtgacatttggtctggcaaaaaacatcaca cctcaaaggcagagtgctttaaatacagaaaagatgtataggacgaattgctgctgc acagagttacagaaacaagttgaaaaacatgaacatattcttctcttcctcttaaaca attcaaccaatcaaaataaatttattgaaatgaaaaaactggtaaaaagttaagtaa gttaaatcgtatgttttcgcctcttctgtgatcaccaataggacatcttcaggcatattg gcaggatagagctaatggagtgaaacctattgtaaggctgtactttcgtgatttaatg acctgaggtttggtcataatgcttctgctgtttttgtaggtttatctgatcgttttcctttg ctactgctaatggaactgaacccccaggggtattccagttgtaatagcctttccttact gttgtttgg 163 221077_at ARMC4 gttgagttgaaattctgccgcttactcaatggccttgggtgatgatgctgtaccctaat tctaaaggaagcaatgaacccccttttcagctaccttactgataagcacttatgttctg ccttctgctatcctgatggttcgggttgtctgtcttactatctacttcttgagtagagag accacattaaatttattgctgtatctcacagggcatcttgctagtgtgcacaggctcgc ctccctacctctgccccgatggtgtgaaggggagagggcgaggttccttagtggcag ggctttgctgttcttcactctcagccccctgaaagcagttcttcctgcctctgagcctgt ctttccttctgctgttaacttctttcctacttttcttgcatccctctcccttccttttcctgcc gtctttcttgtagacat 164 221137_at — aaaaggactaactcacatggctgcagtaagtgctggctgttagctggaagcacaac caaggctgttaacaggtgtgccttggttctcttccatatggcttctcttttgttttcagta ctctgcagtttaattatgatgcatgcaggtgtgaatttctgtttattctgcttgggatgt gttttccttctgggatctgtgaatcggtttctcattatttttgtaaaacctgaagccagtt atctcttaaaataccagctctccttg 165 221168_at PRDM13 ctggacttcttggatgagctcaccctgaaccgcccaggcggtctgctcttggtgttcag aatcacatcaatgcgaacgtcacagcgccttcgagggcgcagattttaactgccacg tatttttaagttgtacttttctgtggaggaaattgtgccttttgaaacgacgttttgtgtg tgtatttcacgttagcatttcattgcataggcaaaacactagtcacaattgggtagat gtgacatccatatacttgtttacattttatctgttctcatgtcaaagactactccttgccc cattgaatatatagtggtagcaggtgtacaaattggtcaagttgcaattatttatgag agaataatgataaatgtaaaatatctaaagcatgaatctaagagcacgcaatatat aattttaaagaaaatattctatttggtagaatacaaatgtggtgtgtgttgttttataat gactgctgtacagtgggtatagtattttggttttggttccagattgtgcaatc 166 221258_s_at KIF18A gtgaagacatcaagagctcgaagtgtaaattacccgaacaagaatcactaccaaat gataacaaagacattttacaacggcttgatccttcttcattctcaactaagcattctat gcctgtaccaagcatggtgccatcctacatggcaatgactactgctgccaaaaggaa acggaaattaacaagttctacatcaaacagttcgttaactgcagacgtaaattctgg atttgccaaacgtgttcgacaagataattcaagtgagaagcacttacaagaaaaca aaccaacaatggaacataaaagaaacatctgtaaaataaatccaagcatggttaga aaatttggaagaaatatttcaaaaggaaatctaagataaatcacttcaaaaccaag caaaatgaagttgatcaaatctgcttttcaaagtttatcaataccctttcaaaaatata tttaaaatctttgaaagaagacccatcttaaagctaagtttacccaagtactttcagc aagc 167 221319_at PCDHB8 cgggagcctgtctcagaactatcagtacgaggtgtgcctggcaggaggctcaggga cgaatgagttccagttcctgaaaccagtattacctaatattcagggccattcttttggg ccagaaatggaacaaaactctaactttaggaatggctttggtttcagccttcagttaa agta 168 221393_at TAAR3 gaactccaccataaagcaactgctggcattttgctggtcagttcctgctcttttttctttt ggtttagttctatctgaggccgatgtttccggtatgcagagctataagatacttgttgc ttgcttcaatttctgtgcccttactttcaacaaattctgggggacaatattgttcactac atgtttctttacccctggctccatcatggttggtatttatggcaaaatctttatcgtttcc aaacagcatgctcgagtcatcagccatgtgcctgaaaacacaaagggggcagtgaa aaaacacctatccaagaaaaaggacaggaaagcagcgaagacactgggtatagta atgggggtgtttctggcttgctggttgccttgttttcttgctgttctgattgacccatacc tagactactccactcccatactaatattggatcttttagtgtggctccggtacttcaact ctacttgcaaccctcttattcatggcttttttaatccatggtttcagaaagcattcaagt acatagtgtcaggaaaaatatttagctcccattcagaaactgc 169 221591_s_at FAM64A cacatctggacccatcagtgactgcctgccatagcctgagagtgtcttggggagacct tgcagagggggagaattgttccttctgctttcctaggggactcttgagcttagaaactc atcgtacacttgaccttgagccttctatttgcctcatctataacatgaagtgctagcat cagatatttgagagctcttagctctgtacccgggtgcctggtttttggggagtcatccg cagagtcactcacccactgtgtttctggtgccaaggctcttgagggccccactctcatc cctcctttccctaccagggactcggaggaaggcataggagatatttccaggcttacg accctgggctcacgggtacctatttatatgctcagtgcagagcactgtggatgtgcca ggaggggtagccctgttcaagagcaatttctgccctttgtaaattatttaagaaacct gctttgtcattttattagaaagaaaccagcgtgtgactttcctagataacactgctttc 170 221609_s_at WNT6 ccgccaggagagcgtgcagctcgaagagaactgcctgtgccgcttccactggtgctg cgtagtacagtgccaccgttgccgtgtgcgcaaggagctcagcctctgcctgtgaccc gccgcccggccgctagactgacttcgcgcagcggtggctcgcacctgtgggacctca gggcaccggcaccgggcgcctctcgccgctcgagcccagcctctccctgccaaagcc caactcccagggctctggaaatggtgaggcgaggggcttgagaggaacgcccaccc acgaaggcccagggcgccagacggccccgaaaaggcgctcggggagcgtttaaag gacactgtacaggccctccctccccttggcctctaggaggaaacagttttttagactg gaaaaaagccagtctaaaggcctctggatactgggctccccagaactgc 171 221718_s_at AKAP13 gcgatgcagaaatgaaccaccggagttcaatgcgagttcttggggatgttgtcagga gacctcccattcataggagaagtttcagtctagaaggcttgacaggaggagctggtg tcggaaacaagccatcctcatctctagaagtaagctctgcaaatgccgaagagctca gacacccattcagtggtgaggaacgggttgactctttggtgtcactttcagaagagga tctggagtcagaccagagagaacataggatgtttgatcagcagatatgtcacagatc taagcagcagggatttaattactgtacatcagccatttcctctccattgacaaaatcc atctcattaatgacaatcagccatcctggattggacaattcacggccctt 172 221950_at EMX2 gtaggctcagcgatagtggtcctcttacagagaaacggggagcaggacgacgggg gngctggggntggcgggggagggtgcccacaaaaagaatcaggacttgtactggg aaaaaaacccctaaattaattatatttcttggacattccctttcctaacatcctgaggc ttaaaaccctgatgcaaacttctcctttcagtggttggagaaattggccgagttcaac cattcactgcaatgcctattccaaactttaaatctatctattgcaaaacctgaaggact gtagttagcggggatgatgttaagtgtggccaagcgcacggcggcaagttttcaagc actgagtttctattccaagatcatagacttactaaagagagtgacaaatgcttcctta atgtcttctataccagaatgtaaatatttttgtgttttgtgttaatttgttagaattctaa cacactatatacttccaa

TABLE 12 Validation of the independent prognostic value of the 15-gene signature in four other separate stage IB-II patient cohorts who received no adjuvant treatment Tumour Hazard Trial/Source Type n Ratio 95% CI p value JBR.10 All NSCLC 62 18.00  5.78-56.05 <0.0001 DCC ADC 96 2.26 1.02-4.97 0.044 NLCI All NSCLC 133 2.27 1.18-4.35 0.014 Duke All NSCLC 48 1.96 0.87-4.42 0.11 UM-SQ SQC 79 3.57 1.48-8.58 0.005 HR: hazard ratio; OBS: observation; NSCLC: non-small cell lung cancer; ADC: adenocarcinoma; SQC: squamous cell carcinoma; DCC Director's Challenge Consortium adenocarcinoma dataset; NLCI: Netherlands Cancer Institute; Duke: Duke University; UM-SQ: University of Michigan, squamous cell carcinoma dataset.

TABLE 13 Demographic features of patients in the four validation sets of stage IB and II patients. Director's Challenge (DCC) All UM HLM MSK NLCI Duke UM-SQ Clinical Factors n = 96 (%) n = 27 (%) n = 38 (%) n = 31 (%) n = 133 (%) n = 48 (%) n = 79 (%) Pathologic subtype Adeno  96 (100)  27 (100)  38 (100)  31 (100) 39 (29) 18 (38) 0 Non-Adeno 0 (0) 0 (0) 0 (0) 0 (0) 94 (71) 30 (62)  79 (100) Stage IB 68 (71) 17 (63) 29 (76) 22 (71) 78 (59) 30 (63) 46 (59) II 28 (29) 10 (37)  9 (24)  9 (29) 55 (41) 18 (37) 33 (41) Age (years)  <65 40 (42) 14 (52) 14 (37) 12 (39) 68 (51) 20 (42) 26 (33) ≧65 56 (58) 13 (48) 24 (63) 19 (61) 65 (49) 28 (58) 53 (67) Sex Male 49 (51) 16 (59) 21 (55) 12 (39) NA 32 (67) 49 (62) Female 47 (49) 11 (41) 17 (45) 19 (61) NA 16 (33) 30 (38) DCC: Directors' Challenge Consortium; UM: University of Michigan; HLM: H Lee Moffitt Cancer Center; MSK: Memorial Sloan-Kettering Cancer Center; NLCI: Netherlands cancer Institute. *Only stage IB-II patients who did not receive adjuvant therapy of any type (chemotherapy or radiotherapy); NA: not available.

TABLE 14 Demographic features of patients in UHN183 validation set (stage I and II) and the training set (BR10-OBS). Clinical factors - A comparative table of the 2 datasets (training and current validation) MedBioGene BR10-OBS N = 183 N = 62 N (%) N (%) Age Median (range) 70 (40-88) 61.2 (35.4-76.7) <65 60 (33) 44 (69) >=65 123 (67) 19 (31) Sex Women 84 (46) 18 (29) Men 99 (54) 44 (71) Stage 1A 49 (27) 1B 80 (44) 34 (55) 2A 9 (5) 28* (45) 2B 45 (25) 3A Histology Adenocarcinoma (ADE) 130 (71) 32 (52) Squamous (SQC) 43 (24) 26 (42) Adenosuamous (ASQ) 2 (1) Large cell (LC) 8 (4) Other 4 (6) 15 gene signature Low risk 90 (49) 29 (47) High risk 93 (51) 33 (53) *Stage 2 or higher

TABLE 15 Best performing custom assays FFPE UHRR/pancreas Assay PCR efficiency (%) R2 PCR efficiency (%) R2 ATP1B1 101.5 0.99 98 0.99 TRIM14 94.5 0.97 107 0.99 FAM64A NA NA 102 0.98 FOSL2 98.5 0.98 95 0.99 HEXIM1 93.5 0.91 99 0.99 MB 103.34 0.91 99.2 0.98 L1CAM 93.92 0.87 87.1 0.8 UMPS 96.5 0.89 97 0.99 EDN3 116 0.82 94.6 0.98 STMN2 102 0.94 104 0.97 IKBKAP 95.5 0.96 97 0.99 MDM2 91.5 0.92 107 0.99 ZNF236 100 −.86 96 0.99

TABLE 16 Primer and Probe Designs SEQ SEQ SEQ ID ID ID AssayID Forward Primer* NO: Reverse Primer* NO: Probe NO: Vendor L1CAM_LNA3 CGGCTACTCTGGAGAGGACTAC 203 CGGCACTTGAGTTGAGGATT 205 (L1CAM-LNA3 #11) Roche (Cat#04685105001) IDT MB_2 AGTTGGTGCTGAACGTCTG 174 GGTGACCCTTAAAGAGCCTG 187 CTGGGATGTCAGCCTCCACCTTC 207 IDT IDT EDN3_2 GTGTCTACTATTGCCACCTGG 175 GCTTCCTCTGTAGTTGGACAG 188 CGTCTGTTCGGGAGTGTTGATCCAAA 208 IDT IDT ATP1B1_1 CCAAATGTCCTTCCCGTTCAG 182 TGCCCAGTCCAAAATACTCC 195 ATCCTTATCTTCATCTCGCTTGCCAG 209 IDT TG IDT FAM64_3 TCCTCAACAGAGCCCCTC 173 TTCTGGAGATGCTGAATTCCC 186 CGAGTCTGACAGTGACCTAGAGCCT 210 IDT FOSL2_LNA3 ACGCCGAGTCCTACTCCA 177 TGAGCCAGGCATATCTACCC 190 (FOSL2-LNA3 #70) Roche (Cat#04688937001) IDT HEXIM1_2 TCGAGGACTCTACTAGCCATG 183 GCTCTTCCTGGACAGCAG 196 TCAACACCAGCCTCAAACTAGCAACT 211 IDT IKBKAP_LNA3 GGAAGCAAGCCCTCTGTGT 184 TCTGCTCAACCAGCTTTCCT 197 (IKBKAP-LNA3 #26) Roche (cat#04687574001) IDT MDM2_2 GAAAACCCCGGATGGTGAG 204 CGAAGCTGGAATCTGTGAGG 206 CAACATGTCTGTACCTACTGATGGTG 212 IDT CT IDT STMN2_1 GAACCTCGCAACATCAACATC 180 GGCTTCAAGATCAGCTCAAAAG 193 AGCAAATCAACAAACGTGCCTCTGG 213 IDT TRIM14_LNA3 CCTCAGCATCCTGAATACATCA 179 TGTGTCAGGTTCCTGTGCTG 192 (TRIM14-LNA3 #7) Roche (cat#04685059001) IDT UMPS_3 ACACAGTGAAAAAGCAGTATGAAG 181 TTTCACAACTCCTGAGCCTG 194 CCACGTGAGCATTTACTAGATCTGCC 214 IDT C IDT ZNF236_3 ACGTAGACCAGTTTGAAGAGC 176 GGCTGCTGATGGTACGTATC 189 TGCTGGTTCGAAGGACTGTTGCG 215 IDT *Sequences in bold are different from what are shown in Table 7.

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1. A method for predicting benefit of adjuvant chemotherapy for a non-small cell lung cancer (NSCLC) patient, comprising: determining a gene expression profile from a sample of the patient's lung tumor, the gene expression profile comprising the level of expression of from 5 to 14 genes each correlative with survival in NSCLC, and being listed in Table 3; and classifying the gene expression profile as being predictive of a positive response to adjuvant chemotherapy or as being predictive of non-responsiveness to adjuvant chemotherapy.
 2. The method of claim 1, wherein the gene expression profile comprises the expression level of from 5 to 13 genes that are correlative with survival in NSCLC, the genes being selected from Table 4A.
 3. The method of claim 1, wherein the gene expression profile does not include expression level of one or both of MLANA and MYT1L.
 4. The method of claim 1, wherein the gene expression profile further comprises the level of expression of one or more normalization genes in the sample.
 5. The method of claim 4, wherein the normalization gene(s) include one or more of BAT1, TBP, PP1A, and GUSB.
 6. The method of claim 5, wherein a normalization gene is GUSB.
 7. The method of claim 1, wherein the gene expression profile is determined by quantitative RT-PCR.
 8. The method of claim 1, wherein the sample is a frozen tumor tissue specimen, or is a formalin-fixed paraffin-embedded tumor tissue specimen.
 9. The method of claim 7, wherein the RT-PCR amplifies at least one target sequence listed in Table
 11. 10. The method of claim 7, wherein the quantitative PCR amplifies at least one target sequence listed in Table
 9. 11. The method of claim 7, wherein the quantitative PCR employs at least one primer or primer set of Table 7 or Table
 16. 12. The method of claim 1, wherein a product of amplification is detected with a probe selected from Table
 16. 13. A method for preparing a gene expression profile indicative of response to adjuvant chemotherapy for non-small cell lung cancer (NSCLC), comprising: determining the level of expression of at least 5 genes from Table 4A in a formalin-fixed paraffin-embedded tumor tissue sample from a lung cancer patient; wherein the expression levels are determined by quantitative RT-PCR, and wherein the gene expression profile includes the expression level of fewer than 15 genes that are correlative with survival in NSCLC. 14-22. (canceled)
 23. The method of claim 1, wherein the tumor tissue sample is from a stage I or stage II NSCLC patient.
 24. A method of prognosing or classifying a subject with non-small cell lung cancer (NSCLC) comprising the steps: a. determining the expression of thirteen biomarkers in a test tumor tissue sample from the subject, wherein the biomarkers correspond to genes in Table 4A, and b. comparing the expression of the thirteen biomarkers in the test sample with expression of the thirteen biomarkers in a control sample, wherein a difference or a similarity in the expression of the thirteen biomarkers between the control and the test sample is used to prognose or classify the subject with NSCLC into a poor survival group or a good survival group.
 25. A method of predicting prognosis in a subject with non-small cell lung cancer (NSCLC) comprising the steps: a. obtaining a subject biomarker expression profile in a tumor tissue sample of the subject; b. obtaining a biomarker reference expression profile associated with a prognosis, wherein the subject biomarker expression profile and the biomarker reference expression profile each have thirteen values, each value representing the expression level of a biomarker, wherein each biomarker corresponds to one gene in Table 4A; and c. selecting the biomarker reference expression profile most similar to the subject biomarker expression profile, to thereby predict a prognosis for the subject.
 26. The method of claim 25, wherein the biomarker reference expression profile comprises a poor survival group or a good survival group. 27-34. (canceled)
 35. A method of selecting a therapy for a subject with NSCLC, comprising the steps: a. determining the expression of thirteen biomarkers in a test tumor tissue sample from the subject, wherein the thirteen biomarkers correspond to the thirteen genes in Table 4A; b. comparing the expression of the thirteen biomarkers in the test sample with the thirteen biomarkers in a control sample; c. classifying the subject in a poor survival group or a good survival group, wherein a difference or a similarity in the expression of the thirteen biomarkers between the control sample and the test sample is used to classify the subject into a poor survival group or a good survival group; d. selecting adjuvant chemotherapy if the subject is classified in the poor survival group and selecting no adjuvant chemotherapy if the subject is classified in the good survival group.
 36. A composition comprising a plurality of isolated nucleic acid sequences, wherein each isolated nucleic acid sequence hybridizes to: a. a RNA product of 13 genes listed in Table 4A; and/or b. a nucleic acid complementary to a), wherein the composition is used to measure the level of RNA expression of the 13 genes. 37-40. (canceled)
 41. A computer implemented product for predicting a prognosis or classifying a subject with NSCLC comprising: a. a means for receiving values corresponding to a subject expression profile in a subject sample; and b. a database comprising a reference expression profile associated with a prognosis, wherein the subject biomarker expression profile and the biomarker reference profile each has thirteen values, each value representing the expression level of a biomarker, wherein each biomarker corresponds to one gene in Table 4A; wherein the computer implemented product selects the biomarker reference expression profile most similar to the subject biomarker expression profile, to thereby predict a prognosis or classify the subject. 42-47. (canceled)
 48. A kit comprising a primer set for amplifying a target sequence in each of from 5 to 14 genes from Table 3, and including at least 5 genes from Table 4A. 49-71. (canceled) 